Methods for modifying plant biomass and cell protectant levels

ABSTRACT

Methods for increasing the biomass of a plant and levels of a cell protectant in a plant, such as sucrose or proline, or plant cell are provided. The methods comprise altering expression levels of a polynucleotide comprising a sequence encoding a C-repeat/DRE binding factor (CBF)-related polypeptide in a plant and identifying plants so altered.. These methods may be used to enhance yield, or cold, freezing, drought, and high salt tolerance of a plant or plant cell.

RELATED APPLICATION INFORMATION

[0001] This application is a continuation-in-part of the following U.S.applications: U.S. application Ser. No. 09/773,990, filed: Feb. 1, 2001,which in turn claimed priority from U.S. application Ser. No.09/580,377, filed: May 26, 2000, which in turn claimed priority fromU.S. provisional Application Serial No. 60/165,860, filed: Nov. 16,1999, now abandoned; U.S. application Ser. No. 09/996,140, filed Nov.26, 2001; U.S. application Ser. No. 09/601,802, filed: Sep. 15, 2000,which in turn claimed priority from PCT application No.: PCT/US99/01895,filed: Jan. 28, 1999, now abandoned, which in turn claimed priority fromU.S. application Ser. No. 09/198,119, filed: Nov. 23, 1998, which issuedas U.S. Pat. No. 6,417,428, which, in turn, claimed priority in part toU.S. application Ser. No. 09/018,233, filed: Feb. 3, 1998, nowabandoned, U.S. application Ser. No. 09/017,816, filed: Feb. 3, 1998,now abandoned, U.S. application Ser. No. 09/018,235, filed: Feb. 3,1998, now abandoned, U.S. application Ser. No. 09/017,575, filed: Feb.3, 1998, now abandoned, U.S. application Ser. No. 09/018,227, filed:Feb. 3, 1998, now abandoned, and U.S. application Ser. No. 09/018,234,filed: Feb. 3, 1998, now abandoned, all six of which claimed priority inpart to U.S. application Ser. No. 08/706,270, filed: Sep. 4, 1996, whichissued as U.S. Pat. No. 5,892,009; U.S. application Ser. No. 09/627,348,filed Jul. 28, 2000, which in turn claimed priority from No. 60/148,200filed Aug. 10, 1999, now abandoned, and U.S. provisional ApplicationSerial No. 60/165,860 filed Nov. 16, 1999, now abandoned; U.S.application Ser. No. 09/713,994, filed: Nov. 16, 2000, which in turnclaimed priority from No. 60/197,899 filed Apr. 17, 2000, now abandoned,U.S. provisional Application Serial No. 60/227,439 filed Aug. 22, 2000,now abandoned, and from U.S. provisional Application Serial No.60/166,228 filed Nov. 17, 1999, now abandoned; each of which areincorporated herein by reference in their entirety.

[0002] This invention was supported by a subcontract under a USDA/CSREESCooperative Agreement and an NSF/SBIR grant. The US Government hascertain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to a method for modifying thebiomass of a plant. The method is particularly useful for modifying theleaf or root biomass of a plant. The invention is also useful formodifying the levels of a cell protectant, such as a cryoprotectant, anosmoprotectant, or the like, in a cell or a plant to improve itsresponse to environmental stresses, including but not limited to cold orfreezing stress, drought stress, or salt stress.

BACKGROUND OF THE INVENTION

[0004] Increasing the biomass of a plant has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. By increasingplant biomass, increased production levels of the products may beobtained from the plants. Tobacco leaves, in particular, have beenemployed as plant factories to generate such products. Furthermore, itmay be desirable to increase crop yields of plants by increasing totalplant photosynthesis. An increase in total plant photosynthesis istypically achieved by increasing leaf area of the plant. Additionalphotosynthetic capacity may be used to increase the yield derived fromparticular plant tissue, including the leaves, roots, fruits or seed. Inaddition, the ability to modify the biomass of the leaves may be usefulfor permitting the growth of a plant under decreased light intensity orunder high light intensity. Modification of the biomass of anothertissue, such as roots, may be useful to improve a plant's ability togrow under harsh environmental conditions, including drought or nutrientdeprivation, because the roots may grow deeper into the ground.

[0005] Due to the commercial consequences of environmental stress damageto crops, there is an interest in understanding how to improve a plant'stolerance to environmental stresses. By improving a plant's performanceor survival in response to different environmental stresses, theweather-related losses in productivity and risks to farming can begreatly reduced. Modifying a plant's tolerance to environmental stressesalso allows a plant to be grown in regions where a plant or plantvariety is typically unable to grow.

[0006] Many biochemical changes occur in a plant when a plant becomestolerant to an environmental stress. For example, for cold or freezingstress tolerance, it is well documented that lipid composition changesoccur during cold acclimation in a wide range of plants and there iscompelling data to indicate that this contributes to greater freezingtolerance (Steponkus et al. (1993) Advances in Low-Temperature Biology,Vol. 2, P. L. Steponkus, editor, (London: JAI Press), pp. 211-312).

[0007] Similarly, the levels of proline and sucrose increase inArabidopsis (McKown et al. (1996) J. Exp. Bot. 47: 1919-1925; Wanner andJunttila (1999) Plant Physiol. 120: 391-400) and other plants (Guy etal. (1992) Plant Physiol. 100: 502-508; Koster and Lynch (1992) PlantPhysiol. 98: 108-113) during cold acclimation and likely have roles infreezing tolerance. There is evidence that proline can protect bothmembranes and proteins against freeze-induced damage in vitro (Rudolphand Crowe (1985) Cryobiol. 22: 367-377; Carpenter and Crowe (1988)Cryobiol. 25: 244-255) and direct evidence that increased levels ofproline enhances whole plant freezing tolerance (Nanjo et al. (1999)FEBS Lett. 461: 205-210).

[0008] Sucrose and other simple sugars have also been shown to beeffective cryoprotectants in vitro (Strauss et al. (1986) Proc. Natl.Acad. Sci. 83: 2422-2426; Carpenter and Crowe (1988) supra) and there iscorrelative evidence indicating a role in freezing tolerance incold-acclimated plants (Guy et al. (1992) supra; Koster and Lynch (1992)supra; Wanner and Junttila (1999) supra).

[0009] Similarly, tolerance to drought or water stress is associatedwith the accumulation of a variety of osmolytes, including sugaralcohols such as mannitol, amino acids such as proline, and glycinebetaine (Greenway et al. (1980) Ann. Rev. Plant Physiol. 31: 149-190;Yancey et al. (1982) Science 217: 1214-1222).

[0010] Cold acclimation in plants is associated with the expression ofcold-regulated (COR) genes that encode many polypeptides of unknownfunction. One of the more intriguing attributes of the COR genes is thatmany encode polypeptides that appear to have biochemical similaritieswith putative cryoprotective proteins (Volger and Heber (1975) Biochim.Biophys. Acta 412: 335-349). Volger and Heber have reported that theleaves of cold-acclimated cabbage and spinach, but not nonacclimatedplants, contain proteins that are effective in protecting isolatedthylakoid membranes against in vitro freeze-thaw damage. Subsequently,Hincha et al. (1990) reported that the cryoprotective proteins act byreducing membrane permeability during freezing and increasing membraneexpandability during thawing (Hincha et al. (1990) Planta 180: 416-419).The biochemical properties of highly enriched fractions of thecryoprotective proteins suggest that the cryoprotective proteins have anumber of properties in common with the polypeptides encoded by many ofthe novel COR genes (Artus et al. (1996) Proc. Natl. Acad. Sci. 93:13404-13409).

[0011] The cold- and drought-regulated COR15a gene of A. thalianaencodes a 15-kDa polypeptide, COR15a, that is targeted to the stromalcompartment of chloroplasts (Lin and Thomashow (1992) Plant Physiol. 99:519-525). During import, COR15a is processed to a mature 9.4-kDapolypeptide, COR15am, that is hydrophilic, remains soluble upon boiling,has a simple amino acid composition (it is rich in both alanine andlysine and devoid of proline, methionine, tryptophan, cysteine,glutamine, arginine, and histidine), is composed largely of a 13-aminoacid motif that is repeated four times, and is predicted to form anamphipathic α-helix. The relatively simple amino acid composition ofCOR15am together with its predicted secondary structure suggests thatthe polypeptide might have a nonenzymatic function and was shown to havethe properties of a cryoprotective protein. (See Artus et al. (1996)supra; Steponkus et al. (1998) Proc. Natl. Acad. Sci. 95: 14570-14575.)

[0012] Thus, the present invention provides a method for modifying theplant biomass by modifying the size or number of leaves or roots of aplant, and for increasing the levels of cell protectants in a cell toallow the cell to tolerate greater environmental stresses.

SUMMARY OF THE INVENTION

[0013] In one aspect, the present invention provides a method formodifying plant biomass and the level of a cell protectant in a cell ora plant. The method comprises transforming a plant cell or plant with arecombinant polynucleotide comprising a sequence encoding a C-repeat/DREbinding factor (CBF)-related polypeptide and expressing the CBF-relatedpolypeptide in the plant cell. Expression of the CBF-related polypeptidemodifies biomass and the level of the cell protectant in the cell orplant. The method may optionally comprise cold-acclimating the cell toincrease the levels of cell protectants in the transformed cell or planteven further.

[0014] In one embodiment, the recombinant polynucleotide encodes aCBF-related polypeptide comprising the AP2 domain comprising amino acids45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81,83-91, 93-96, 99, 101, 102, and 104-106 of CBF1 (SEQ ID NO: 2). In asecond embodiment, the recombinant polynucleotide encodes a CBF-relatedpolypeptide comprising a CBF-related polypeptide and comprises one ormore of the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQID NO: 320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent anyamino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO.2). In a third embodiment, the recombinant polynucleotide encodes aCBF-related polypeptide comprising a CBF-related polypeptide andcomprises one or more of the following peptides: PKKXAGR (SEQ ID NO:322; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any aminoacid) or AGRKXF (SEQ ID NO: 324; amino acids 35-40 of SEQ ID NO. 2; ‘X’may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids42-46 of SEQ ID NO. 2). In a fourth embodiment, the recombinantpolynucleotide encodes a CBF-related polypeptide comprising aCBF-related polypeptide and comprises one or more of the followingpeptides: PKKRAGR (SEQ ID NO: 323; amino acids 31-37 of SEQ ID NO. 2) orAGRKKF (SEQ ID NO: 325; amino acids 35-40 of SEQ ID NO. 2) or ETRHP (SEQID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a fifth embodiment,the recombinant polynucleotide encodes a CBF-related polypeptidecomprising a CBF-related polypeptide and comprises one or more of thefollowing peptides: PKKPAGR (SEQ ID NO: 326; amino acids 31-37 of SEQ IDNO. 2) or AGRKKF (SEQ ID NO: 325; amino acids 35-40 of SEQ ID NO. 2) orETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In anotherembodiment, the recombinant polynucleotide encodes a CBF-relatedpolypeptide comprising SEQ ID NO:15. In yet another embodiment, therecombinant polynucleotide comprises SEQ ID NO:14.

[0015] Additionally, the recombinant polynucleotide may comprise aregulatory region operably linked to the sequence encoding theCBF-related polypeptide. The regulatory region may comprise aconstitutive promoter, an inducible promoter, a tissue specific promoteror a developmental stage specific promoter. The leaf or root biomass ofa plant may be modified. Cell protectants whose levels may be modifiedinclude proline, sugars, such as sucrose, or lipids, such as fattyacids, or cryoprotective proteins. As a result of the increased levelsof any of these cell protectants, or of a combination of any these cellprotectants, the environmental stress tolerance of a cell is increased.The environmental stresses may be cold or freezing tolerance, droughttolerance or high salinity tolerance.

[0016] In a second aspect, the present invention is another method formodifying the biomass of a plant. This method comprises altering thelevels of a polynucleotide comprising a sequence encoding a C-repeat/DREbinding factor (CBF)-related polypeptide in a plant and identifying aplant so modified. The altered polynucleotide expression modifies thebiomass of the plant.

[0017] In another aspect, the present invention is a method forimproving the tolerance of a cell or plant to an environmental stress.The method comprises transforming the cell or plant with a recombinantpolynucleotide comprising a sequence encoding a C-repeat/DRE bindingfactor (CBF)-related polypeptide and expressing the CBF-relatedpolypeptide in the transformed cell or plant. Expression of theCBF-related polypeptide typically increases cell protectant levels atleast 1.5 fold in the transformed cell or plant compared with cellprotectant levels in an untransformed cell or plant. The enhanced cellprotectant levels improve the environmental stress tolerance of the cellor plant.

[0018] In one embodiment, the recombinant polynucleotide encodes aCBF-related polypeptide comprising the AP2 domain comprising amino acids45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81,83-91, 93-96, 99, 101, 102 and 104-106 of CBF1 (SEQ ID NO: 2). In asecond embodiment, the CBF-related polypeptide may comprise one or moreof the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids 31-37 ofSEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQ ID NO:320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any aminoacid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In athird embodiment, the recombinant polynucleotide encodes a CBF-relatedpolypeptide comprising a CBF-related polypeptide and comprises one ormore of the following peptides: PKKXAGR (SEQ ID NO: 322; amino acids31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRKXF (SEQID NO: 324; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent anyamino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO.2). In a fourth embodiment, the recombinant polynucleotide encodes aCBF-related polypeptide comprising a CBF-related polypeptide andcomprises one or more of the following peptides: PKKKAGR (SEQ ID NO:323; amino acids 31-37 of SEQ ID NO. 2) or AGRKKF (SEQ ID NO: 325; aminoacids 35-40 of SEQ ID NO. 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46of SEQ ID NO. 2). In another embodiment, the recombinant polynucleotideencodes a CBF-related polypeptide comprising SEQ ID NO:15. In yetanother embodiment, the recombinant polynucleotide comprises SEQ IDNO:14.

[0019] Additionally, the recombinant polynucleotide may comprise aregulatory region operably linked to the sequence encoding theCBF-related polypeptide. The regulatory region may comprise aconstitutive promoter, an inducible promoter, a tissue specific promoteror a developmental stage specific promoter. Cell protectants whoselevels may be modified include proline, sugars, such as sucrose, orlipids, such as fatty acids, or crypoprotective proteins. As a result ofthe increased levels of any of these cell protectants or the combinationof any of these cell protectants, the environmental stress tolerance ofa cell or plant is improved.

[0020] In a further aspect, the present invention is a method forproducing a cell protectant. The method comprises transforming a cell orplant with a recombinant polynucleotide comprising a sequence encoding aC-repeat/DRE binding factor (CBF)-related polypeptide, expressing saidrecombinant polypeptide in the transformed cell so as to increase thelevels of the cell protectant in the cell or plant, and then isolatingthe cell protectant from the transformed cell or plant. In oneembodiment, the recombinant polynucleotide encodes a CBF-relatedpolypeptide comprising SEQ ID NO:15. In another embodiment, therecombinant polynucleotide comprises SEQ ID NO:14.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

[0021] The file of this patent contains at least one drawing executed inclor. Copies of this patent with clor drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

[0022] The Sequence Listing provides exemplary polynucleotide andpolypeptide sequences of the invention. The traits associated with theuse of the sequences are included in the Examples.

[0023]FIGS. 1A and 1B show how the yeast reporter strains wereconstructed.

[0024]FIG. 1A is a schematic diagram showing the screening strategy.

[0025]FIG. 1B is a chart showing activity of the positive cDNA clones inyeast reporter strains.

[0026]FIGS. 2A, 2B, 2C and 2D provide an analysis of the pACT-11 cDNAclone.

[0027]FIG. 2A is a schematic drawing of the pACT-11 cDNA insertindicating the location and 5′ to 3′ orientation of the 24 kDapolypeptide and 25s rRNA sequences.

[0028]FIG. 2B is a DNA and amino acid sequence of the 24 kDa polypeptide(SEQ ID NO: 1 and SEQ ID NO: 2).

[0029]FIG. 2C is a schematic drawing indicating the relative positionsof the potential nuclear localization signal (NLS), the AP2 domain andthe acidic region of the 24 kDa polypeptide.

[0030]FIG. 2D is a chart showing comparison of the AP2 domain of the 24kDa polypeptide (SEQ ID NO:10) with that of the tobacco DNA bindingprotein EREBP2 (SEQ ID NO:11).

[0031]FIG. 3 is a chart showing activation of reporter genes by the 24kDa polypeptide.

[0032]FIG. 4 is a photograph of an electrophoresis gel showingexpression of the recombinant 24 kDa polypeptide in E. coli.

[0033]FIG. 5 is a photograph of a gel for shift assays indicating thatCBF1 binds to the C-repeat/DRE.

[0034]FIG. 6 is a photograph of a Southern blot analysis indicating CBF1is a unique or low copy number gene.

[0035]FIGS. 7A, 7B and 7C relate to CBF1 transcripts in control andcold-treated Arabidopsis plants.

[0036]FIG. 7A is a photograph of a Northern blot analysis of RNAisolated from Arabidopsis plants that were grown at 22° C. (0 h) orgrown at 22° C. and transferred to 2.5° C. for the indicated times (2,4, 8, or 24 h).

[0037]FIG. 7B is a graph showing relative transcript levels of CBF1 incontrol and cold-treated plants.

[0038]FIG. 7C is a graph showing relative transcript levels of COR15a incontrol and cold-treated plants.

[0039]FIG. 8 is a Northern blot showing CBF1 and COR transcript levelsin RLD and transgenic Arabidopsis plants.

[0040]FIG. 9 is an immunoblot showing COR15am protein levels in RLD andtransgenic Arabidopsis plants.

[0041]FIGS. 10A and 11B are graphs showing freezing tolerance of leavesfrom RLD and transgenic Arabidopsis plants.

[0042]FIG. 11 is a photograph showing freezing survival of RLD and A6Arabidopsis plants.

[0043]FIG. 12 shows the DNA sequence for CBF2 (SEQ ID NO:12) encodingthe polypeptide sequence CBF2 (SEQ ID NO:13).

[0044]FIG. 13 shows the DNA sequence for CBF3 (SEQ ID NO:14) encodingthe polypeptide sequence CBF3 (SEQ ID NO:15).

[0045]FIG. 14 shows the amino acid alignment of proteins CBF1 (SEQ IDNO:2), CBF2 (SEQ ID NO:13), and CBF3 (SEQ ID NO:15).

[0046]FIG. 15 is a graph showing induction of reporter genes in yeastthat carry the C-repeat/DRE regulatory element CBF1, CBF2, or CBF3.

[0047]FIG. 16 shows the amino acid sequence of a portion of a canola CBFhomolog (SEQ ID NO:17) and its alignment to the amino acid sequence ofCBF1 (SEQ ID NO:2).

[0048]FIGS. 17A, 17B, 17C, 17D, 17E, 17F and 17G show restriction mapsof plasmids pMB12008, pMB12009, pMB12010, pMB12011, pMB12012, pMB12013,and pMB12014, respectively.

[0049]FIG. 18A shows the DNA sequences for the CBF homologs fromBrassica juncea, Brassica napus, Brassica oleracea, Brassica rapa,Glycine max, Raphanus sativus, and Zea mays.

[0050]FIG. 18B shows the amino acid sequences (one-letter abbreviations)encoded by the DNA sequences (shown in FIG. 18A) for CBF homologs fromBrassica juncea, Brassica napus, Brassica oleracea, Brassica rapa,Glycine max, Raphanus sativus, and Zea mays.

[0051]FIG. 19A shows an amino acid alignment of the AP2 domains ofseveral CBF proteins (SEQ ID NOs: 137 through 167) with the consensussequence between the proteins highlighted as well as a comparison of theAP2 domains with that of the tobacco DNA binding protein EREBp2 (SEQ IDNO: 168).

[0052]FIG. 19B shows an amino acid alignment of the AP2 domains ofseveral CBF proteins (SEQ ID NOs: 137 through 167) including dreb2a (SEQID NO: 169) and dreb2b (SEQ ID NO: 170) with the consensus sequencebetween the proteins highlighted.

[0053]FIG. 19C shows an amino acid alignment of the AP2 domains ofseveral CBF proteins (SEQ ID NOs: 137 through 167) including dreb2a (SEQID NO: 169), dreb2b (SEQ ID NO: 170), and tiny (SEQ ID NO: 171) with theconsensus sequence between the proteins highlighted.

[0054]FIG. 19D shows a difference between the consensus sequence shownin FIG. 19A (SEQ ID NOs: 137 through 167) and tiny (SEQ ID NO: 171).

[0055]FIG. 19E shows a difference between the consensus sequence shownin FIG. 19B (SEQ ID NOs: 137 through 167) and tiny (SEQ ID NO: 171).

[0056]FIG. 20 shows an amino acid alignment of the amino terminus ofseveral CBF proteins (SEQ ID NOs: 172 through 194) with their consensussequence highlighted.

[0057]FIG. 21A (SEQ ID NOs: 195 through 266) and FIG. 21B (SEQ ID NOs:267 through 293) show an amino acid alignment of the carboxy terminus ofseveral CBF proteins, with their consensus sequences highlighted.

[0058]FIG. 22 shows the effect of CBF3 expression on proline levels.Free proline levels were determined in leaf tissue from control Ws-2 andB6 plants and CBF3-expressing A40, A30 and A28 plants grown at 20° C.(warm) or plants grown at 20° C. and cold-treated at 5° C. for 7 days (7d cold).

[0059]FIG. 23 shows the effect of CBF3 expression on transcript levelsof genes involved in proline and sugar metabolism. Northern analysis oftotal RNA (20 μg for CBF3; 5 μg for other genes) isolated from controlArabidopsis Ws-2 and B6 plants and from CBF3-expressing A40, A30 and A28plants. Plants were grown at 20° C., then cold-treated at 5° C. for thetimes indicated. The blots were hybridized with probes for CBF3, COR78,P5CS2, Suc synthase (SuSy), Suc-phosphate synthase (SPS), and elF4a, aconstitutively expressed gene used as a loading control (Metz et al.Gene 120: 313 (1992)).

[0060]FIG. 24 shows the effect of CBF3 Expression on levels of totalsoluble sugars. Total soluble sugars were determined for leaf tissuefrom control Ws-2 and B6 plants and CBF3-expressing A40, A30 and A28plants grown at 20° C. (warm) or plants grown at 20° C. and cold-treatedat 5° C. for 7 days (7 d cold).

[0061]FIG. 25 shows the effect of CBF3 expression on fatty acidcomposition. The fatty acid profiles of total lipids extracted from leaftissue of control Ws-2 plants and CBF3-expressing A28 plants weredetermined for plants grown at 20° C. (unfilled bars) or at 20° C.followed by 7 days at 5° C. (filled bars).

[0062]FIG. 26 shows the effect of CBF3 expression on freezing tolerance.(A) Seedlings of Ws-2 and A30 were grown at 20° C. on solid medium andfrozen at −2° C. for 24 hours followed by 24 hours at −6+ C.; (B)Control Ws-2 and CBF3-expressing transgenic A40, A30 and A28 plants weregrown at 20° C. and the freezing tolerance of leaves was measured usingthe electrolyte leakage test; (C and D) Same as (B) except that plantswere grown at 20° C. followed by 7 days cold acclimation at 5° C.

[0063] FIGS. 27A-C show a comparison of leaf number to bolting time (A),biomass to bolting time (B) and days to bolt (C) for Arabidopsisthaliana plants overexpressing a CBF-related polypeptide (13-4, 2-1,16-3, 16-2, 17-10) compared with a wild-type plant (wt-control).

[0064]FIG. 28 shows a comparison of the leaves of two plants oneoverexpressing a polynucleotide encoding a CBF-related polypeptide (A)and a second plant not overexpressing the polynucleotide (B).

[0065]FIG. 29 shows a comparison of the roots of two plants oneoverexpressing a polynucleotide encoding a CBF-related polypeptide (A)and a second plant not overexpressing the polynucleotide (B).

[0066]FIG. 30 shows a comparison of the % electrolyte leakage for aplant overexpressing CBF3 (SEQ ID NO: 15) (A40a2, A28a3, A28a3) or acontrol plant (WS-2), none of which have been cold acclimated.

[0067]FIG. 31 shows a comparison of the % electrolyte leakage for aplant overexpressing CBF3 (SEQ ID NO: 15) (A28a3, A30a1) or a controlplant (WS-2) after cold acclimation.

[0068]FIG. 32 shows a comparison of the % electrolyte leakage for aplant overexpressing CBF2 (SEQ ID NO: 13) (E24, E8 or E2) or a controlplant (WS-2, B6), none of which have been cold acclimated.

[0069]FIG. 33 shows a comparison of the % electrolyte leakage for aplant overexpressing CBF2 (SEQ ID NO: 13) (E24, E8 or E2) or a controlplant (WS-2, B6), after cold acclimation.

[0070]FIGS. 34A through 34D shows an alignment of CBF sequences fromArabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from Medicago trunculata(SEQ ID NOs: 294 through 300). Amino acid residues with identity betweenaligned sequences are boxed. The consensus sequence is shown below eachset of alignments.

[0071]FIGS. 35A through 35D shows an alignment of CBF sequences fromArabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from Oryza sativa (SEQID NOs: 301 through 310). Amino acid residues with identity betweenaligned sequences are boxed. The consensus sequence is shown below eachset of alignments.

[0072]FIGS. 36A through 36C shows an alignment of CBF sequences fromArabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from Zea mays (SEQ IDNOs: 311 through 315). Amino acid residues with identity between alignedsequences are boxed. The consensus sequence is shown below each set ofalignments.

[0073]FIG. 37 shows that CBFs from Medicago trunculata, Oryza sativa,and Arabidopsis (CBF3) activate transcription from the Arabidopsis CORgene promoter from the RD29a gene, as measured by fold-increase overcontrol (pMEN65 plasmid control=1 unit). The CBF sequences are:Arabidopsis CBF3 (SEQ ID NO:15; CBF3), Medicago trunculata CBF (SEQ IDNO:294; mt G3362), Medicago trunculata CBF (SEQ ID NO:295; mt G3364),Medicago trunculata CBF (SEQ ID NO:296; mt G3365), Medicago trunculataCBF (SEQ ID NO:297; mt G3366), Medicago trunculata CBF (SEQ ID NO:298;mt G3367), Medicago trunculata CBF (SEQ ID NO:299; mt G3368), Medicagotrunculata CBF (SEQ ID NO:300; mt G3369), Oryza sativa CBF (SEQ IDNO:301; os G3370), Oryza sativa CBF (SEQ ID NO:302; os G3371), Oryzasativa CBF (SEQ ID NO:303; os G3372), Oryza sativa CBF (SEQ ID NO:304;os G3373), Oryza sativa CBF (SEQ ID NO:308; os G3377), Oryza sativa CBF(SEQ ID NO:309; os G3378), and Oryza sativa CBF (SEQ ID NO:310; osG3379).

DETAILED DESCRIPTION OF THE INVENTION

[0074] Definitions

[0075] “Environmental stress tolerance” refers to a decrease in theextent of a cell's injury or growth inhibition or an increase insurvival rate after exposure to cold or freezing temperatures, droughtconditions, high salinity environments or the like.

[0076] A “cell protectant” refers to a compound that improves theenvironmental stress tolerance of a cell. The cell protectant may be acryoprotectant or an osmoprotectant. The cell protectant may be prolineor any metabolically related compound, sugars or any metabolicallyrelated compound, and a variety of lipids, including fatty acids, whichprotect a cell's integrity during an environmental stress.

[0077] A “polynucleotide” is a nucleotide sequence comprising a genecoding sequence or a fragment thereof (comprising at least 18consecutive nucleotides, preferably at least 30 consecutive nucleotides,and more preferably at least 50 consecutive nucleotides). Additionally,the polynucleotide may comprise a promoter, an intron, an enhancerregion, a polyadenylation site, a translation initiation site, 5′ or 3′untranslated regions, a reporter gene, a selectable marker or the like.The polynucleotide may comprise single stranded or double stranded DNAor RNA. The polynucleotide may comprise modified bases or a modifiedbackbone. The polynucleotide may be genomic, a transcript (such as anmRNA) or a processed nucleotide sequence (such as a cDNA). Thepolynucleotide may comprise a sequence in either sense or antisenseorientations.

[0078] A “recombinant polynucleotide” is a polynucleotide that is not inits native state, for example, the polynucleotide is comprised of anucleotide sequence not found in nature or the polynucleotide isseparated from nucleotide sequences with which it typically is inproximity or is next to nucleotide sequences with which it typically isnot in proximity or is expressed at different levels.

[0079] A “consensus sequence”, with regard to nucleotide sequences,refers to a nucleotide sequence that serves to represent a family ofsimilar, experimentally-derived sequences. Each position in theconsensus sequence is assigned a base that corresponds to the mostfrequently occurring nucleotide in the experimentally-derived sequences,when sequences are compared in an alignment. A “consensus sequence”,with regard to polypeptide sequences, refers to a polypeptide sequencethat serves to represent a family of similar, experimentally-derivedsequences. Each position in the consensus sequence is assigned an aminoacid residue that corresponds to the most frequently occurring aminoacid residue in the experimentally-derived sequences, when sequences arecompared in an alignment.

[0080] A “transformed plant” refers to a plant that contains geneticmaterial not normally found in a wild type plant and which has beenintroduced into a plant by human manipulation. A transformed plant is aplant that may contain an expression vector or cassette. The expressioncassette comprises a gene coding sequence and allows for the expressionof the gene coding sequence. The expression cassette may be introducedinto a plant by transformation or by breeding after transformation of aparent cell. In particular, the transformed plant may refer to a wholeplant as well as to a plant part, such as flower, seed, fruit, leaf, orroot, plant tissue, plant cells or any other plant material, and progenythereof.

[0081] A “transformed cell” refers to a cell that contains geneticmaterial not normally found in a wild type cell and which has beenintroduced into the cell by human manipulation. A transformed cell is acell that may contain an expression vector or cassette. The expressioncassette comprises a gene coding sequence and allows for the expressionof the gene coding sequence. The expression cassette may be introducedinto a cell by transformation or by breeding after transformation of aparent cell. A transformed cell may refer to a cell from any organism,including mammalian cells, plant cells, bacterial cells, and the like.In particular, the transformed cell is a plant cell and may refer to awhole plant as well as to a plant part, such as seed, fruit, leaf, orroot, plant tissue, plant cells or any other plant material, and progenythereof.

[0082] The term “modified expression” in reference to polynucleotide orpolypeptide expression refers to an expression pattern in a transformedcell or plant that is different from the expression pattern in the wildtype cell or plant; for example, by expression in a cell type or planttissue other than a cell type or plant tissue in which thepolynucleotide or polypeptide is naturally expressed, or by expressionat a time other than at the time the polynucleotide or polypeptide isexpressed in the wild type cell or plant, or by a response to differentinducible agents, such as hormones or environmental signals, or atdifferent expression levels (either higher or lower) compared to thoseobserved a wild type cell or plant. The term may also refer to loweringthe levels of expression to below the detection level or completelyabolishing expression. The resulting expression pattern may be transientor stable, constitutive or inducible.

[0083] A “CBF-related polypeptide” or “CBF” is a protein transcriptionfactor that binds to a promoter comprising a cold- anddehydration-responsive DNA regulatory element known as the CRT(C-repeat)/DRE (dehydration responsive element) (Baker et al. (1994) iPlant. Mol. Biol. 24: 701-713; Yamaguchi-Shinozaki and Shinozaki (1994)Plant Cell 6: 251-264). The terms “CBF-related polypeptide” and “CBF”are interchangeable. These proteins comprise an AP2/EREBP DNA bindingmotif (Riechmann and Meyerowitz (1998) Biol. Chem. 379: 633-646) and aretranscription factors (Stockinger et al. (1997) Proc. Natl. Acad. Sci.94: 1035-1040). The AP2 domain, which may also be referred to as the ERFdomain (Hao et al. (1998) J. Biol. Chem 273: 26857-26861) may compriseamino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73,75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of CBF1 (G40; SEQID NO: 2). Additionally, the CBF-related polypeptide may comprise one ormore of the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQID NO: 320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent anyamino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO:2).

[0084] A “regulatory region” is a region that can regulate thetranscription of a gene coding sequence. The regulatory region may be apromoter or an enhancer. The regulatory region sequence is “operablylinked” when it is placed into a functional relationship with the genecoding sequence. For example, a promoter or enhancer is operably linkedto a gene coding sequence if the presence of the promoter or enhancerincreases the level of expression of the gene coding sequence. Promotersmay increase transcription of a gene at all times (constitutivepromoter), increase transcription only in the presence of specificagents or events (inducible promoter), increase transcription inspecific tissue(s) (tissue specific promoter) or during specific stagesof cell or tissue or organism development (developmental stage specificpromoter).

[0085] “Plant biomass modification” refers to a detectable difference inthe size of one or more plant tissues, such as the leaves, roots, stem,fruit or flowers of a transformed plant expressing a polynucleotide orpolypeptide of the present invention compared with a plant not doing so.The modification may entail at least a 5% increase or decrease in anobserved trait (difference), at least a 10% difference, at least a 20%difference, at least a 30%, at least a 50%, at least a 70%, at least a100% or a greater difference. It is known that there may be a naturalvariation in plant biomass. Therefore, the modification observed entailsa change in the normal distribution of the trait in transformed plantscompared with the distribution observed in wild type plants.

[0086] “Cell protectant level modification” refers to a detectabledifference in cell protectant levels in a transformed cell expressing apolynucleotide or polypeptide of the present invention compared with acell not doing so, such as a wild type cell. The trait modification mayentail at least a 5% increase or decrease in an observed trait(difference), at least a 10% difference, at least a 20% difference, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater difference. It is known that there may be a natural variation inthe modified cell protectant levels. Therefore, the cell protectantlevel modification observed entails a change in the normal distributionof the levels in transformed cells compared with the distributionobserved in wild type cells.

[0087] “Cold-acclimating a cell” and “cold acclimation” refers to aprocess whereby a transformed cell is exposed to temperatures belowabout 12° C. for different periods of time to elicit higher levels of acell protectant compared with cell protectant levels in a cell that hasnot been cold acclimated.

[0088] Traits that May Be Modified

[0089] Trait modifications of particular interest include those to seed(such as embryo or endosperm), fruit, root, flower, leaf, stem, shoot,seedling or the like, including: enhanced tolerance to environmentalconditions including freezing, chilling, heat, drought, watersaturation, radiation and ozone; improved tolerance to microbial, fungalor viral diseases; improved tolerance to pest infestations, includingnematodes, mollicutes, parasitic higher plants or the like; decreasedherbicide sensitivity; improved tolerance of heavy metals or enhancedability to take up heavy metals; improved growth under poorphotoconditions (for example, low light and/or short day length), orchanges in expression levels of genes of interest. Other phenotype thatcan be modified relate to the production of plant metabolites, such asvariations in the production of taxol, tocopherol, tocotrienol, sterols,phytosterols, vitamins, wax monomers, anti-oxidants, amino acids,lignins, cellulose, tannins, prenyllipids (such as chlorophylls andcarotenoids), glucosinolates, and terpenoids, enhanced orcompositionally altered protein or oil production (especially in seeds),or modified sugar (insoluble or soluble) and/or starch composition.Physical plant characteristics or traits that can be modified includecell development (such as the number of trichomes), fruit and seed sizeand number, yields of plant parts such as stems, leaves, inflorescences,and roots, the stability of the seeds during storage, characteristics ofthe seed pod (for example, susceptibility to shattering), root hairlength and quantity, internode distances, or the quality of seed coat.Plant growth characteristics that can be modified include growth rate,germination rate of seeds, vigor of plants and seedlings, leaf andflower senescence, male sterility, apomixis, flowering time, flowerabscission, rate of nitrogen uptake, osmotic sensitivity to solublesugar concentrations, biomass or transpiration characteristics, as wellas plant architecture characteristics such as apical dominance,branching patterns, number of organs, organ identity, organ shape orsize.

[0090] Transcription Factors Modify Expression of Endogenous Genes

[0091] Expression of genes that encode transcription factors that modifyexpression of endogenous genes, polynucleotides, and proteins are wellknown in the art. In addition, transgenic plants comprising isolatedpolynucleotides encoding transcription factors may also modifyexpression of endogenous genes, polynucleotides, and proteins. Examplesinclude Peng et al. (1997) Genes and Development 11: 3194-3205) and Penget al. (1999) Nature 400: 256-261). In addition, many others havedemonstrated that an Arabidopsis transcription factor expressed in anexogenous plant species elicits the same or very similar phenotypicresponse. See, for example, Fu et al. (2001) Plant Cell 13: 1791-1802);Nandi et al. (2000) Curr. Biol. 10: 215-218); Coupland (1995) Nature377: 482-483); and Weigel et al. (1995) Nature 377: 482-500).

[0092] In another example, Mandel et al. (1992) Cell 71: 133-143) andSuzuki et al. (2001) i Plant J. 28: 409-418) teach that a transcriptionfactor expressed in another plant species elicits the same or verysimilar phenotypic response of the endogenous sequence, as oftenpredicted in earlier studies of Arabidopsis transcription factors inArabidopsis. Other examples can be found in the teachings of Müller etal. (2001, Plant J. 28: 169-179); Kim et al. (2001) Plant J. 25:247-259); Kyozuka and Shimamoto (2002) Plant Cell Physiol. 43: 130-135);Boss and Thomas (2002) Nature, 416: 847-850); He et al. (2000)Transgenic Res. 9: 223-227); and Robson et al. (2001) Plant J. 28:619-631).

[0093] In yet another example, Gilmour et al. (1998) Plant J. 16:433-442) and Jaglo-Ottosen et al. (1998) Science 280: 104-106) teach anArabidopsis AP2 transcription factor, CBF1, which, when overexpressed intransgenic plants, increases plant freezing tolerance. An alignment ofthe CBF proteins from Arabidopsis, B. napus, l wheat, rye, and tomatorevealed the presence of conserved amino acid sequences,PKK/RPAGRxKFxETRHP and DSAWR, that flank the AP2/EREBP DNA bindingdomains of the proteins and distinguish them from other members of theAP2/EREBP protein family.

[0094] Polypeptides and Polynucleotides of the Invention

[0095] The present invention provides, among other things, transcriptionfactors (TFs), and transcription factor homolog polypeptides, andisolated or recombinant polynucleotides encoding the polypeptides, ornovel variant polypeptides or polynucleotides encoding novel variants oftranscription factors derived from the specific sequences provided here.These polypeptides and polynucleotides may be employed to modify aplant's characteristic.

[0096] Exemplary polynucleotides encoding the polypeptides of theinvention were identified by screening an Arabidopsis cDNA expressionlibrary for clones encoding C-repeat/DRE binding domains (Stockinger etal. (1997) Proc. Natl. Acad. Sci. 94: 1035-1040). The cDNA libraryharbored in Escherichia coli BNN132 was amplified, and plasmid DNA wasisolated and transformed into yeast GGY1 reporter strains. CBF geneswere expressed in E. coli and yeast, and gel shift assays, in whichorientation and concatenation number of inserts were determined bydideoxy DNA sequence analysis, were conducted to confirm that the CBFgene products bound to the C-repeat/DRE (Stockinger (1997) supra).Sequences initially identified were then further characterized toidentify sequences comprising specified sequence strings correspondingto sequence motifs present in families of known transcription factors.In addition, further exemplary polynucleotides encoding the polypeptidesof the invention were identified in the plant GenBank database usingpublicly available sequence analysis programs and parameters. Sequencesinitially identified were then further characterized to identifysequences comprising specified sequence strings corresponding tosequence motifs present in families of known transcription factors.Polynucleotide sequences meeting such criteria were confirmed astranscription factors.

[0097] Additional polynucleotides of the invention were identified byscreening Arabidopsis thaliana and/or other plant cDNA libraries withprobes corresponding to known transcription factors under low stringencyhybridization conditions. Additional sequences, including full lengthcoding sequences were subsequently recovered by the rapid amplificationof cDNA ends (RACE) procedure, using a commercially available kitaccording to the manufacturer's instructions. Where necessary, multiplerounds of RACE are performed to isolate 5′ and 3′ ends. The full-lengthcDNA was then recovered by a routine end-to-end polymerase chainreaction (PCR) using primers specific to the isolated 5′ and 3′ ends.Exemplary sequences are provided in the Sequence Listing.

[0098] The polynucleotides of the invention can be or were ectopicallyexpressed in overexpressor or knockout plants and the changes in thecharacteristic(s) or trait(s)characteristics or traits of the plantsobserved. Therefore, the polynucleotides and polypeptides can beemployed to improve the characteristics or traits of plants.

[0099] The polynucleotides of the invention can be or were ectopicallyexpressed in overexpressor plant cells and the changes in the expressionlevels of a number of genes, polynucleotides, and/or proteins of theplant cells observed. Therefore, the polynucleotides and polypeptidescan be employed to change expression levels of a genes, polynucleotides,and/or proteins of plants.

[0100] Producing Polypeptides

[0101] The polynucleotides of the invention include sequences thatencode transcription factors and transcription factor homologpolypeptides and sequences complementary thereto, as well as uniquefragments of coding sequence, or sequence complementary thereto. Suchpolynucleotides can be, for example, DNA or RNA, for example, mRNA,cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides,etc. The polynucleotides are either double-stranded or single-stranded,and include either, or both sense (i.e., coding) sequences and antisense(i.e., non-coding, complementary) sequences. The polynucleotides includethe coding sequence of a transcription factor, or transcription factorhomolog polypeptide, in isolation, in combination with additional codingsequences (for example, a purification tag, a localization signal, as afusion-protein, as a pre-protein, or the like), in combination withnon-coding sequences (for example, introns or inteins, regulatoryelements such as promoters, enhancers, terminators, and the like),and/or in a vector or host environment in which the polynucleotideencoding a transcription factor or transcription factor homologpolypeptide is an endogenous or exogenous gene.

[0102] A variety of methods exist for producing the polynucleotides ofthe invention. Procedures for identifying and isolating DNA clones arewell known to those of skill in the art, and are described in, forexample, Berger and Kimmel, Guide to Molecular Cloning TechniquesMethods in Enzymology volume 152; Academic Press, Inc., San Diego,Calif. (“Berger”); Sambrook et al. Molecular Cloning—A Laboratory Manual(2nd edition), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., editors, Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., (supplemented through 2000) (“Ausubel”).

[0103] Alternatively, polynucleotides of the invention, can be producedby a variety of in vitro amplification methods adapted to the presentinvention by appropriate selection of specific or degenerate primers.Examples of protocols sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification andother RNA polymerase mediated techniques (for example, NASBA), forexample, for the production of the homologous nucleic acids of theinvention are found in Berger (supra), Sambrook (supra) and Ausubel(supra), as well as Mullis et al. (1987) PCR Protocols A Guide toMethods and Applications, Innis et al., editors, (1990) Academic PressInc. San Diego, Calif. (“Innis”). Improved methods for cloning in vitroamplified nucleic acids are described in Wallace et al. U.S. Pat. No.5,426,039. Improved methods for amplifying large nucleic acids by PCRare summarized in Cheng et al. (1994) Nature 369: 684-685 and thereferences cited therein, in which PCR amplicons of up to 40 kb aregenerated. One of skill will appreciate that essentially any RNA can beconverted into a double stranded DNA suitable for restriction digestion,PCR expansion and sequencing using reverse transcriptase and apolymerase. See, for example, Ausubel, Sambrook and Berger, all supra.

[0104] Alternatively, polynucleotides and oligonucleotides of theinvention can be assembled from fragments produced by solid-phasesynthesis methods. Typically, fragments of up to approximately 100 basesare individually synthesized and then enzymatically or chemicallyligated to produce a desired sequence, for example, a polynucleotideencoding all or part of a transcription factor. For example, chemicalsynthesis using the phosphoramidite method is described, for example, byBeaucage et al. (1981) Tetrahedron Letters 22: 1859-1869; and Matthes etal. (1984) EMBO J. 3: 801-805. According to such methods,oligonucleotides are synthesized, purified, annealed to theircomplementary strand, ligated and then optionally cloned into suitablevectors. And if so desired, the polynucleotides and polypeptides of theinvention can be custom ordered from any of a number of commercialsuppliers.

[0105] Homologous Sequences

[0106] Sequences homologous, i.e., that share significant sequenceidentity or similarity, to those provided in the Sequence Listing,derived from Arabidopsis thaliana or from other plants of choice arealso an aspect of the invention. Homologous sequences can be derivedfrom any plant including monocots and dicots and in particularagriculturally important plant species, including but not limited to,crops such as soybean, wheat, corn, potato, cotton, rice, rape, oilseedrape (including rapeseed and canola), sunflower, alfalfa, sugarcane andturf; or fruits and vegetables, such as banana, blackberry, blueberry,strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee,cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion,papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn,tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach,pear, cherry and plum) and vegetable brassicas (such as broccoli,cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops,fruits and vegetables whose phenotype can be changed include barley,rye, millet, sorghum, currant, avocado, citrus fruits such as oranges,lemons, grapefruit and tangerines, artichoke, cherries, nuts such as thewalnut and peanut, endive, leek, roots, such as arrowroot, beet,cassava, turnip, radish, yam, and sweet potato, and beans. Thehomologous sequences may also be derived from woody species, such pine,poplar and eucalyptus, or mint or other labiates.

[0107] Orthologs and Paralogs

[0108] Several different methods are known by those of skill in the artfor identifying and defining these functionally homologous sequences.Three general methods for defining paralogs and orthologs are described;a paralog or ortholog or homolog may be identified by one or more of themethods described below.

[0109] Orthologs and paralogs are evolutionarily related genes that havesimilar sequence and similar functions. Orthologs are structurallyrelated genes in different species that are derived by a speciationevent. Paralogs are structurally related genes within a single speciesand that are derived by a duplication event.

[0110] Within a single plant species, gene duplication may cause twocopies of a particular gene, giving rise to two or more genes withsimilar sequence and similar function known as paralogs. A paralog istherefore a similar gene with a similar function within the samespecies. Paralogs typically cluster together or in the same clade (agroup of similar genes) when a gene family phylogeny is analyzed usingprograms such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680; Higgins et al. (1996) Methods Enzymol. 266 383-402). Groupsof similar genes can also be identified with pair-wise BLAST analysis(Feng et al. (1987) J. Mol. Evol. 25: 351-360). For example, a clade ofvery similar MADS domain transcription factors from Arabidopsis allshare a common function in flowering time (Ratcliffe et al. (2001) PlantPhysiol. 126: 122-132), and a group of very similar AP2 domaintranscription factors from Arabidopsis are involved in tolerance ofplants to freezing (Gilmour et al. (1998) supra). Analysis of groups ofsimilar genes with similar function that fall within one clade can yieldsub-sequences that are particular to the lade. These sub-sequences,known as consensus sequences, can not only be used to define thesequences within each clade, but define the functions of these genes;genes within a clade may contain paralogous sequences, or orthologoussequences that share the same function. (See also, for example, Mount,D. W. (2001) Bioinformatics: Sequence and Genome Analysis Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., page 543.)

[0111] Speciation, the production of new species from a parentalspecies, can also give rise to two or more genes with similar sequenceand similar function. These genes, termed orthologs, often have anidentical function within their host plants and are ofteninterchangeable between species without losing function. Because plantshave common ancestors, many genes in any plant species will have acorresponding orthologous gene in another plant species. Once aphylogenic tree for a gene family of one species has been constructedusing a program such as CLUSTAL (Thompson et al. (1994) Nucleic AcidsRes. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266:383-402), potential orthologous sequences can placed into thephylogenetic tree and its relationship to genes from the species ofinterest can be determined. Once the ortholog pair has been identified,the function of the test ortholog can be determined by determining thefunction of the reference ortholog.

[0112] Transcription factors that are homologous to the listed sequenceswill typically share at least about 30% amino acid sequence identity, orat least about 30% amino acid sequence identity outside of a knownconsensus sequence or consensus DNA-binding site. More closely relatedtranscription factors can share at least about 50%, about 60%, about65%, about 70%, about 75% or about 80% or about 90% or about 95% orabout 98% or more sequence identity with the listed sequences, or withthe listed sequences but excluding or outside a known consensus sequenceor consensus DNA-binding site, or with the listed sequences excludingone or all conserved domain. Factors that are most closely related tothe listed sequences share, for example, at least about 85%, about 90%or about 95% or more % sequence identity to the listed sequences, or tothe listed sequences but excluding or outside a known consensus sequenceor consensus DNA-binding site or outside one or all conserved domain. Atthe nucleotide level, the sequences will typically share at least about40% nucleotide sequence identity, preferably at least about 50%, about60%, about 70% or about 80% sequence identity, and more preferably about85%, about 90%, about 95% or about 97% or more sequence identity to oneor more of the listed sequences, or to a listed sequence but excludingor outside a known consensus sequence or consensus DNA-binding site, oroutside one or all conserved domain. The degeneracy of the genetic codeenables major variations in the nucleotide sequence of a polynucleotidewhile maintaining the amino acid sequence of the encoded protein.Conserved domains within a transcription factor family may exhibit ahigher degree of sequence homology, such as at least 65% sequenceidentity including conservative substitutions, and preferably at least80% sequence identity, and more preferably at least 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 90%, or at least about 95%, or at least about 98% sequenceidentity. Transcription factors that are homologous to the listedsequences should share at least 30%, or at least about 60%, or at leastabout 75%, or at least about 80%, or at least about 90%, or at leastabout 95% amino acid sequence identity over the entire length of thepolypeptide or the homolog. In addition, transcription factors that arehomologous to the listed sequences should share at least 30%, or atleast about 60%, or at least about 75%, or at least about 80%, or atleast about 90%, or at least about 95% amino acid sequence similarityover the entire length of the polypeptide or the homolog.

[0113] Percent identity can be determined electronically, for example,by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). TheMEGALIGN program can create alignments between two or more sequencesaccording to different methods, for example, the clustal method (see,for example, Higgins et al. (1988) Gene 73: 237-244). The clustalalgorithm groups sequences into clusters by examining the distancesbetween all pairs. The clusters are aligned pairwise and then in groups.Other alignment algorithms or programs may be used, including FASTA,BLAST, or ENTREZ, FASTA and BLAST. These are available as a part of theGCG sequence analysis package (University of Wisconsin, Madison, Wis.),and can be used with or without default settings. ENTREZ is availablethrough the National Center for Biotechnology Information. In oneembodiment, the percent identity of two sequences can be determined bythe GCG program with a gap weight of 1, for example, each amino acid gapis weighted as if it were a single amino acid or nucleotide mismatchbetween the two sequences (see U.S. Pat. No. 6,262,333).

[0114] Other techniques for alignment are described in Methods inEnzymology, vol. 266: Computer Methods for Macromolecular SequenceAnalysis (1996), editor, Doolittle, Academic Press, Inc., San Diego,Calif. Preferably, an alignment program that permits gaps in thesequence is utilized to align the sequences. The Smith-Waterman is onetype of algorithm that permits gaps in sequence alignments. See MethodsMol. Biol. 70: 173-187 (1997). Also, the CLUSTALW alignment algorithm(for example, in the MACVECTOR 6.0 or MACVECTOR 6.5 applications,Accelrys, San Diego Calif.) may be utilized to align sequences. Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. An alternative search strategy uses MPSRCHsoftware, which runs on a MASPAR computer. MPSRCH uses a Smith-Watermanalgorithm to score sequences on a massively parallel computer. Thisapproach improves ability to pick up distantly related matches, and isespecially tolerant of small gaps and nucleotide sequence errors.Nucleic acid-encoded amino acid sequences can be used to search bothprotein and DNA databases.

[0115] The percentage similarity between two polypeptide sequences, forexample, sequence A and sequence B, is calculated by dividing the lengthof sequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweenpolynucleotide sequences can also be counted or calculated by othermethods known in the art, for example, the Jotun Hein method (see, forexample, Hein (1990) Methods Enzymol. 183: 626-645). Identity betweensequences can also be determined by other methods known in the art, forexample, by varying hybridization conditions (see U.S. patentapplication No. 20010010913).

[0116] Thus, the invention provides methods for identifying a sequencesimilar or paralogous or orthologous or homologous to one or morepolynucleotides as noted herein, or one or more target polypeptidesencoded by the polynucleotides, or otherwise noted herein and mayinclude linking or associating a given plant phenotype or gene functionwith a sequence. In the methods, a sequence database is provided(locally or across an inter or intra net) and a query is made againstthe sequence database using the relevant sequences herein and associatedplant phenotypes or gene functions.

[0117] In addition, one or more polynucleotide sequences or one or morepolypeptides encoded by the polynucleotide sequences may be used tosearch against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25:217-221), PFAM, and other databases which contain previously identifiedand annotated motifs, sequences and gene functions. Methods that searchfor primary sequence patterns with secondary structure gap penalties(Smith et al. (1992) Protein Eng. 5: 35-51) as well as algorithms suchas Basic Local Alignment Search Tool (BLAST; Altschul, S. F. (1993) J.Mol. Evol. 36:290-300; Altschul et al. (1990) supra), BLOCKS (Henikoffand Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden MarkovModels (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammeret al. (1997) Proteins 28: 405-420), and the like, can be used tomanipulate and analyze polynucleotide and polypeptide sequences encodedby polynucleotides. These databases, algorithms and other methods arewell known in the art and are described in Ausubel et al. (1997; ShortProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit7.7) and in Meyers (1995; Molecular Biology and Biotechnology, WileyVCH, New York N.Y., pages 856-853).

[0118] Furthermore, methods using manual alignment of sequences similaror homologous to one or more polynucleotide sequences or one or morepolypeptides encoded by the polynucleotide sequences may be used toidentify regions of similarity and conserved domains. Such manualmethods are well-known of those of skill in the art and can include, forexample, comparisons of tertiary structure between a polypeptidesequence encoded by a polynucleotide which comprises a known functionwith a polypeptide sequence encoded by a polynucleotide sequence whichhas a function not yet determined. Such examples of tertiary structuremay comprise predicted alpha helices, beta-sheets, amphipathic helices,leucine zipper motifs, zinc finger motifs, proline-rich regions,cysteine repeat motifs, and the like.

[0119] CBF Genes and Related Sequences

[0120] Many plants, including Arabidopsis, show increased resistance tofreezing after they have been exposed to low, non-freezing temperatures.This cold-acclimation response is associated with the induction of COR(cold-regulated) genes mediated by the C-repeat/drought-responsiveelement (CRT/DRE) DNA regulatory element) (Baker et al. (1994) Plant.Mol. Biol. 24: 701-713; Yamaguchi-Shinozaki and Shinozaki (1994) PlantCell 6: 251-264). Increased expression of Arabidopsis CBF genes(transcriptional activators that bind to the CRT/DRE sequence), induceCOR gene expression and increase the freezing tolerance of non-coldacclimated Arabidopsis plants. CBF genes are thus regulators of the coldacclimation response, and act by controlling the level of COR geneexpression, which in turn promotes tolerance to freezing. CBF genes havealso been shown to influence cell protectant levels such as proline inplants, which can lead to increased plant biomass (Kishor et al. (1995)Plant Physiol. 108: 1387-1394).

[0121] It is believed that a significant class of environmental stresstolerance and biomass regulatory genes encode for binding proteins withan AP2 domain capable of binding to a DNA regulatory sequence. Each ofthe presently disclosed CBF gene sequences encodes a binding proteinthat includes an AP2 domain, the latter being a DNA-binding motifsimilar to those present in Arabidopsis proteins APETALA2, AINTEGUMENTAand TINY, the tobacco ethylene response element binding proteins, andnumerous other plant proteins. The AP2 domains of CBF binding proteinsin general, including the CBF binding proteins described herein, sharesignificant homology, and comprise a consensus sequence sufficientlyhomologous to any one of the consensus sequences shown in FIG. 19A, 19B,or 19C that the binding protein is capable of binding to a CCGregulatory sequence, preferably a CCGAC regulatory sequence.Specifically, CBF proteins comprise an AP2/EREBP DNA binding motif(Riechmann and Meyerowitz (1998) Biol. Chem. 379: 633-646) and aretranscription factors (Stockinger et al. (1997) supra). The AP2 domainof CBF proteins may comprise amino acids 45, 46, 48, 50-52, 54, 59, 60,62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and104-106 of CBF1 (G40; SEQ ID NO: 2), or the consensus sequence:

[0122] H P X_(n) Y X_(n) G V R X_(n) R X_(n) W V X_(n) E X_(n) R E X_(n)N K X_(n) R I W X_(n) G T F X_(n) T X_(n) E X_(n) A A R A H D V A AX_(n) A L R G X_(n) A X_(n) L N X_(n) A D S

[0123] where X is any amino acid residue and n is any number of aminoacid residues.

[0124] CBF1 (G40; SEQ ID NO: 2), CBF2 (G41; SEQ ID NO: 13) and CBF3(G42; SEQ ID NO: 15) have similar sequences, particularly in the AP2domain (defined by the consensus sequence defined above and bounded by:HP X_(n) Y X_(n) GVR X_(n) ADS). CBF2 has 95% sequence identity withCBF1 in the AP2 domain, and CBF3 shares 96% sequence identity with CBF1in the AP2 domain. These three and related genes can be used to preparetransgenic plants and plants with altered traits. CBF1, for example, wasstudied using transgenic plants in which the gene encoding the proteinwas expressed under the control of the 35S promoter (for a more completediscussion of studies involving CBF1, CBF2 and CBF3 experiments, seeexamples below). CBF1 was shown to improve tolerance to freezing andsalt stress in Arabidopsis and Canola. CBF1 could thus be used tomanipulate those tolerances, and to generate plants that might germinateand survive under such adverse conditions. For example, evaporation fromthe soil surface causes upward water movement and salt accumulation inthe upper soil layer where the seeds are placed. Thus, germinationnormally takes place at a salt concentration much higher than the meansalt concentration in the whole soil profile. Increased salt toleranceduring the germination stage of a crop plant would impact survivabilityand yield. If the activity of CBF1 is regulated at a post-translationallevel (for example, by being phosphorylated), it might be possible toengineer constitutively active versions of the protein that protect theplant under adverse environmental conditions As seen in FIGS. 27Athrough 27C, CBF1-overexpressing plants grew more slowly, than thecontrol plants. However, as the transgenic plants flowered later theygrew for a longer period of time prior to bolting; at this developmentalstage the transgenic plants had more leaves and higher biomass than thecontrol plants at the same developmental stage (which the control plantreached a few days earlier).

[0125] Similarly, overexpression of CBF3 in transformed plants increasedthe time to flowering; the plants grew for a longer period of time priorto bolting and increased the number of rosette leaves per plant (seeExample 22, below). Thus, CBF polypeptide overexpression could be usedto manipulate time to flowering, leaf number, and plant biomass. If theactivity of CBF polypeptides is regulated at a post-translational level(for example, by being phosphorylated), it might be possible to engineerconstitutively active versions of the protein that increase leaf numberand/or biomass.

[0126] G912 (CBF4; SEQ ID NO: 97). G912 was recognized by Applicants asthe AP2/EREBP gene most closely related to Arabidopsis CBF1, CBF2, andCBF3 (SEQ ID NO: 2, 13, and 15, respectively) (Haake et al. (2002) PlantPhysiol. 130: 639-648; Stockinger et al. (1997) supra); Gilmour et al.(1998) supra), G912 shares 93%, 91 and 93% sequence identity with CBF1,CBF2 and CBF3, respectively, in the AP2 domain. G912 sequence similaritywith CBF1, 2 and 3 extends beyond the conserved AP2 domain. ThisAP2/EREBP transcription factor is also closely related to the members ofthe CBF-like subgroup of AP2/EREBP proteins from other plants, such asAF084185 Brassica napus dehydration responsive element binding protein.G912 was identified in the sequence of P1 clone MSG15 (GenBank accessionnumber AB015478; gene MSG15.6; no published information is availableabout the functions of G912).

[0127] G912 expression appears to be induced by cold, drought, andosmotic stress. The function of G912 was studied using transgenic plantsin which this gene was expressed under the control of the 35S promoter.As with plants overexpressing CBF1, CBF2 and CBF3, plants overexpressingG912 were dark green, and flowered later than non-transformed controlplants. Plants overexpressing G912 were more freezing and droughttolerant than the wild-type controls.

[0128] All these results mirror the extensive body of work presentedherein that has shown that related genes CBF1, CBF2, and CBF3 areinvolved in the control of the low-temperature response in Arabidopsis,and that those genes can be used to improve freezing, drought, and salttolerance in plants (Stockinger et al. (1997) supra; Gilmour et al.(1998) supra; Jaglo-Ottosen et al. (1998) Science 280: 104-106; Liu etal. (1998) Plant Cell 10: 1391-1406, Kasuga et al. (1999) NatureBiotechnol. 17: 287-291). In addition, G912 overexpressing plants alsoexhibit a sugar sensing phenotype: i.e., reduced seedling vigor andcotyledon expansion upon germination on high glucose media.

[0129] Polypeptide Transcription Factors from other Plant Species (OddNumbered SEQ ID NO: 39-95 and Even numbered 116-128).

[0130] As described in more detail in Example 15, below, a PCR strategywas used to isolate CBF homologs from a number of species of plants bothrelated and diverse from Arabidopsis. These species included Brassicajuncea, Brassica napus, Brassica oleracea, Brassica rapa, Glycine max,Raphanus sativus, Secale cereale, Triticum aestivum, and Zea mays. Thenucleotide (for example, bjCBF1) and peptide sequences (for example,BJCBF 1-PEP) of these isolated CBF homologs are shown in FIGS. 18A and18B, respectively. Table 11 (which may be found in Example 15) lists thesequence names and SEQ ID NO: of these isolated CBF homologs. Thepercentage sequence identity of each of the AP2 domains of the sequencesfrom other species with the Arabadopsis CBF1 AP2 domain (subsequence ofG40, SEQ ID NO: 2) is also shown and is 80% identity for the Zea maysAP2 domain and from 85-93% for the other six species.

[0131] SEQ ID NOs: 39-45 are from B. juncea. The AP2 regions present ineach of these sequences, and which may be found as subsequences in thecorresponding sequences in the Sequence Listing, are:

[0132] SEQ ID NO: 39 (percent sequence identity with CBF1: 87% (50/57)):PGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRAACLNFADS

[0133] SEQ ID NO: 143 (percent sequence identity with CBF1: 85%(53/62)): HPIYRGVRLRKSGKWVCEVREPNKRSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS

[0134] SEQ ID NO: 145 (percent sequence identity with CBF1: 85%(53/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFLTAEIAARAHDVAAIALRGKSACLNFADS

[0135] SEQ ID NO: 140 (percent sequence identity with CBF1: 93%(58/62)): HPIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFADS

[0136] SEQ ID NOs: 47-63 are from B. napus. The AP2 regions present ineach of these sequences are:

[0137] SEQ ID NO: 148 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0138] SEQ ID NO: 146 (percent sequence identity with CBF1: 87%(54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0139] SEQ ID NO: 147 (percent sequence identity with CBF1: 87%(54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0140] SEQ ID NO: 153 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0141] SEQ ID NO: 154 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0142] SEQ ID NO: 149 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0143] SEQ ID NO: 144 (percent sequence identity with CBF1: 87%(54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS

[0144] SEQ ID NO: 150 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0145] SEQ ID NO: 165 (percent sequence identity with CBF1: 85%(53/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGARLNYADS

[0146] SEQ ID NOs: 65-73 are from B. oleracea. The AP2 regions presentin each of these sequences are:

[0147] SEQ ID NO: 163 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRAACLNFADS

[0148] SEQ ID NO: 141 (percent sequence identity with CBF1: 87%(54/62)): HPVYRGVRLRNSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS

[0149] SEQ ID NO: 151 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0150] SEQ ID NO: 155 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0151] SEQ ID NO: 164 (percent sequence identity with CBF1: 87%(54/62)): HPIYRGVRLRKSGKWVCEVRELNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0152] SEQ ID NOs: 75-87 are from B. rapa. The AP2 regions present ineach of these sequences are:

[0153] SEQ ID NO: 156 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0154] SEQ ID NO: 152 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS

[0155] SEQ ID NO: 157 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0156] SEQ ID NO: 158 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0157] SEQ ID NO: 159 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0158] SEQ ID NO: 160 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0159] SEQ ID NO: 161 (percent sequence identity with CBF1: 88%(55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0160] SEQ ID NO: 89 is from Glycine max. The AP2 region present in thissequence is:

[0161] SEQ ID NO: 167 (percent sequence identity with CBF1: 87%):HPIYSGVRRRNTDKWVSEVREPNKKTRIWLGTFPTPEMAARAHDVAAMALRGRYACLNFADS

[0162] SEQ ID NO: 91-93 are from Raphanus sativus. The AP2 regionspresent in each of these sequences are:

[0163] SEQ ID NO: 162 (percent sequence identity with CBF1:HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS

[0164] SEQ ID NO: 142 (percent sequence identity with CBF1: 88%(55/62)):

[0165] HPIYRGVRLRNSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS

[0166] SEQ ID NO: 95 is from Zea mays. The AP2 region present in thissequence is:

[0167] SEQ ID NO: 166 (percent sequence identity with CBF1: 80%(51/63)):HPVYRGVRRRGPAGRWVCEVREPNKKSRIWLGTFATPEAAARAHDVAALALRGRAACLNFADS

[0168] SEQ ID NO: 116-124 are from Secale cereale. The AP2 regionspresent in each of these sequences are:

[0169] SEQ ID NO: 116 (percent sequence identity with CBF1: 71% (37/52))ATAQDGEVGAAGRWVCEVRVLGMRGSRLWLGTFVTAEMAARAHDAAVLALSGRKACLNFADS

[0170] SEQ ID NO: 118 (percent sequence identity with CBF1: 72% (46/64))HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSCRAACLNFADS

[0171] SEQ ID NO: 120 (percent sequence identity with CBF1: 73% (47/64))HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSGRKACLNFADS

[0172] SEQ ID NO: 122 (percent sequence identity with CBF1: 69%(44/64))HPLYRGVRRRGRLGQWVCEVRVRGAQGYRLWLGTFTTAEMAARAHDSAVLALLDRAACLNFADS

[0173] SEQ ID NO: 124 (percent sequence identity with CBF1: (73% (47/64)HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSGRAACLNFADS

[0174] SEQ ID NO: 126 is from Triticum aestivum. The AP2 region presentin this sequence is:

[0175] SEQ ID NO: 126 (percent sequence identity with CBF1: (73% (47/64)HPLYRGVRRRGRVGQWVCEVRVPGVKGSRLWLGTFTTAEMAARAHDAAVLALSGRAACLNFADS

[0176] SEQ ID NO: 128 is found in Glycine max. The AP2 region present inthis sequence is:

[0177] SEQ ID NO: 128 (percent sequence identity with CBF1: (89% (57/64)HPVYRGVRRRNSDKWVCEVREPNKKTRIWLGTFPTPEMAARAHDVAAMALRGRYACLNFADS

[0178] Other Arabidopsis Homologs

[0179] G2513 (SEQ ID NO: 99) G2513 is also closely related to CBF1,CBF2, and CBF3 (SEQ ID NO: 2, 13, and 15, respectively). G2513 shares73% sequence identity with CBF1, 73% sequence identity with CBF2, and52%% sequence identity with CBF3. In the AP2 domain G2513 shares 77%,75%, and 80% identity with CBF1, CBF2 and CBF3, respectively.

[0180] G2513 corresponds to gene T12C24.14 (AAF88096). G2513 showssequence similarity, outside of the conserved AP2 domain, with a proteinfrom Nicotiana tabacum (gi12003384 AF211531_(—)1 Avr9/Cf-9 rapidlyelicited protein 111B [Nicotiana tabacum]). No published information isavailable about the functions of G2513. G2513-overexpressing plants wereinitially small with narrow dark green leaves, grew slowly and initiatedfloral buds several weeks later than in wild-type controls.

[0181] G2513 forms part of a monophyletic group within the ArabidopsisAP2/EREBP family that also includes G40 (SEQ ID NO: 2), G41 (SEQ ID NO:13), G41 (SEQ ID NO: 15), and G912 (SEQ ID NO: 97), (CBF1, CBF2, CBF3and CBF4, respectively). However, the clade is divided into twosubgroups, one comprised by the four CBF genes (SEQ ID NOs: 2, 13, 15,and 97) and the other by G2107 (SEQ ID NO:101) and G2513 (SEQ ID NO:99).

[0182] G2513 is ubiquitously expressed, at significantly higher levelsin rosette leaves, flower, and embryo tissues. Because of itsphylogenetic relationship to the CBF genes and the delay in floral buddevelopment, G2513 may be used to delay flowering and increase plantbiomass relative to control after the plant flowers.

[0183] G2107 (SEQ ID NO: 101) shares 44% sequence identity with CBF1(SEQ ID NO: 2), 45% sequence identity with CBF2 (SEQ ID NO: 13), and51%% sequence identity with CBF3 (SEQ ID NO: 15). In the AP2 domainG2107 shares 75%, 74%, and 79% sequence identity with CBF1, CBF2, andCBF3, respectively. G2107 shows sequence similarity, outside of theconserved AP2 domain, with a protein from Nicotiana tabacum (gi12003384AF211531_(—)1 Avr9/Cf-9 rapidly elicited protein 111B [Nicotianatabacum]). G2107 corresponds to gene F16M19.17 (AAF18701). No publishedinformation is available about the function of G2107.

[0184] G2107 expression is detected in floral tissues (including embryoand silique), as well as in rosette leaves, but not in roots orgerminating seedlings. Because of its phylogenetic relationship to theCBF genes and the delay in floral bud development, G2107 may be used todelay flowering and increase plant biomass relative to control after thelatter flowers or when environmental conditions induce stress in controlplants.

[0185] G21 (SEQ ID NO: 103) corresponds to gene At2g44940 (AAD32841).G21 corresponds to gene At2g44940 (AAD32841). G2107 shares 57% sequenceidentity with CBF1 (SEQ ID NO: 2), 58% sequence identity with CBF2 (SEQID NO: 13), and 53%% sequence identity with CBF3 (SEQ ID NO: 15). In theAP2 domain G21 shares 76%, 72%, and 74% sequence identity with CBF1,CBF2, and CBF3, respectively. G21 does not show extensive sequencesimilarity with known genes from other plant species outside of theconserved AP2/EREBP domain.

[0186] Overexpression of G21 caused alterations in plant growth anddevelopment: 35S::G21 plants were smaller than wild type, oftenpossessed curled, darker green leaves, and showed reduced fertility. Noalterations were detected in 35S::G21 plants in the physiological andbiochemical analyses that were performed.

[0187] G21 is ubiquitously expressed, and appears to be induced byseveral environmental or physiological conditions, in particular coldand abscisic acid. Because of its phylogenetic relationship to the CBFgenes and the delay in floral bud development, G21 may also be used todelay flowering and increase plant biomass relative to control after thelatter flowers, or when environmental conditions induce stress incontrol plants.

[0188] Summing up the presently disclosed means by which the levels of acell protectant in a plant cell or plant may be modified, it has beenshown that:

[0189] a) CBF1, CBF2, CBF3 are expressed in response to environmentalstresses. For example, CBF1 expression increases in response to coldstress.

[0190] b) CBF1, CBF2, CBF3 have been shown to modify the levels of cellprotectants in plant cells. These cell protectants include proline,sugars and fatty acids.

[0191] c) CBF1, CBF2, CBF3 have been shown to confer tolerance toenvironmental stresses. In Arabidopsis, for example, overexpression ofany of these polypeptides results in improved tolerance to cold, highsalt and drought.

[0192] d) A variety of plant genera and species may be transformed witha recombinant polynucleotide encoding a C-repeat/DRE binding factor(CBF)-related polypeptide; these species include Arabidopsis thaliana,leaf mustard (Brassica juncea), Brassica oleracea (including cabbage,Brussels sprouts, broccoli, kohlrabi, cauliflower, and kale), Brassicarapa (including turnip greens, turnip rape, and field mustard), rapeseedand canola (Brassica rapa, Brassica campestris L., and Brassica napusL), Brassica napus (in addition to rapeseed and canola, also includesrutabaga and Swedish turnip), soybean (Glycine max), radish and cloverradish (Raphanus sativus), corn (Zea mays), wheat (Triticum), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor andSorghum vulgare, and barley (Hordeum vulgare).

[0193] e) Different plants may be made more tolerant to environmentalstresses. Overexpression of the paralogous genes CBF1, CBF2, and CBF3 indifferent plants, including Arabidopsis and canola, resulted inincreased tolerance to several environmental stresses, includingfreezing, salt, and drought tolerance.

[0194] f) Orthologous sequences to CBF genes have been identified incanola, soybeans, rice, corn and other diverse plant species. Thisdemonstrates that CBF genes are present and likely function in a similarmanner in diverse species.

[0195] g) Overexpression of paralogs of the CBF genes, including CBF4(G912), have also been shown to confer improved tolerance toenvironmental stress, which demonstrates that genes encodingpolypeptides with the AP2 domain or a similarly functioning variant areable to confer improved stress resistance.

[0196] h) Gao et al. ((2002) Plant Mol. Biol. 49 (5), 459-471) havecharacterized four CBF transcription factors from Brassica napus thatfunction in a similar manner and show a high degree of sequencesimilarity to Arabidopsis CBF.

[0197] i) Zhou et al. have identified a dehydration responsive elementbinding protein GenBank Accession No. AAD45623) that shares 88% identitywith the conserved domain of CBF1.

[0198] j) Durrant et al. (2002; unpublished; GenBank Accession No.AAG43548) have identified Avr9/Cf-9 (rapidly elicited protein 111B) inNicotiana tabacum, the cDNA expression profiling of which reveals rapid,resistance gene-dependent, active oxygen-independent, gene inductionduring the plant defense response; this protein shares 64% identity withthe CBF1 sequence and 85% (53/62 residues) with the conserved domain ofCBF1.

[0199] Identifying Polynucleotides or Nucleic Acids by Hybridization

[0200] Polynucleotides homologous to the sequences illustrated in theSequence Listing and tables can be identified, for example, byhybridization to each other under stringent or under highly stringentconditions. Single stranded polynucleotides hybridize when theyassociate based on a variety of well characterized physical-chemicalforces, such as hydrogen bonding, solvent exclusion, base stacking andthe like. The stringency of a hybridization reflects the degree ofsequence identity of the nucleic acids involved, such that the higherthe stringency, the more similar are the two polynucleotide strands.Stringency is influenced by a variety of factors, including temperature,salt concentration and composition, organic and non-organic additives,solvents, etc. present in both the hybridization and wash solutions andincubations (and number thereof), as described in more detail in thereferences cited above.

[0201] Encompassed by the invention are polynucleotide sequences thatare capable of hybridizing to the claimed transcription factorpolynucleotide sequences, and, in particular, to those shown in SEQ IDNO: 1, 12, 14, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115,117, 119, 121, 123, 125, and 127, and fragments thereof under variousconditions of stringency. (See, for example, Wahl et al. (1987) MethodsEnzymol. 152: 399-407; Kimmel (1987) Methods Enzymol. 152: 507-511.) Inaddition to the nucleotide sequences listed in Tables 4 and 5, fulllength cDNA, orthologs, and paralogs of the present nucleotide sequencesmay be identified and isolated using well-known methods. The cDNAlibraries orthologs, and paralogs of the present nucleotide sequencesmay be screened using hybridization methods to determine their utilityas hybridization target or amplification probes.

[0202] With regard to hybridization, conditions that are highlystringent, and means for achieving them, are well known in the art. See,for example, Sambrook et al. (1989) “Molecular Cloning: A LaboratoryManual” (2nd ed., Cold Spring Harbor Laboratory); Berger and Kimmel,editors, (1987) “Guide to Molecular Cloning Techniques”, In Methods inEnzymology: 152: 467-469; and Anderson and Young (1985) “QuantitativeFilter Hybridisation.” In: Hames and Higgins, editors, Nucleic AcidHybridisation, A Practical Approach. Oxford, IRL Press, pages 73-111.

[0203] Stability of DNA duplexes is affected by such factors as basecomposition, length, and degree of base pair mismatch. Hybridizationconditions may be adjusted to allow DNAs of different sequencerelatedness to hybridize. The melting temperature (T_(m)) is defined asthe temperature when 50% of the duplex molecules have dissociated intotheir constituent single strands. The melting temperature of a perfectlymatched duplex, where the hybridization buffer contains formamide as adenaturing agent, may be estimated by the following equations:

[0204] (I) DNA-DNA:

T_(m)(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−0.62(% formamide−500/L

[0205] (II) DNA-RNA:

T_(m)(° C.)=79.8+18.5(log[Na+])+0.58(% G+C)+0.12(% G+C)²−0.5(%formamide)−820/L

[0206] (III) RNA-RNA:

T_(m)(° C.)=79.8+18.5(log[Na+])+0.58(% G+C)+0.12(% G+C)²−0.35(%formamide)−820/L

[0207] where L is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, and % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, approximately 1° C. is requiredto reduce the melting temperature for each 1% mismatch.

[0208] Hybridization experiments are generally conducted in a buffer ofpH between 6.8 to 7.4, although the rate of hybridization is nearlyindependent of pH at ionic strengths likely to be used in thehybridization buffer (Anderson et al. (1985) supra). In addition, one ormore of the following may be used to reduce non-specific hybridization:sonicated salmon sperm DNA or another non-complementary DNA, bovineserum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS),polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfateand polyethylene glycol 6000 act to exclude DNA from solution, thusraising the effective probe DNA concentration and the hybridizationsignal within a given unit of time. In some instances, conditions ofeven greater stringency may be desirable or required to reducenon-specific and/or background hybridization. These conditions may becreated with the use of higher temperature, lower ionic strength andhigher concentration of a denaturing agent such as formamide.

[0209] Stringency conditions can be adjusted to screen for moderatelysimilar fragments such as homologous sequences from distantly relatedorganisms, or to highly similar fragments such as genes that duplicatefunctional enzymes from closely related organisms. The stringency can beadjusted either during the hybridization step or in thepost-hybridization washes. Salt concentration, formamide concentration,hybridization temperature and probe lengths are variables that can beused to alter stringency (as described by the formula above). As ageneral guidelines high stringency is typically performed at T_(m)-5° C.to T_(m)-20° C., moderate stringency at T_(m)-20° C. to T_(m)-35° C. andlow stringency at T_(m)-35° C. to T_(m)-50° C. for duplex >150 basepairs. Hybridization may be performed at low to moderate stringency(25-50° C. below T_(m)), followed by post-hybridization washes atincreasing stringencies. Maximum rates of hybridization in solution aredetermined empirically to occur at T_(m)-25° C. for DNA-DNA duplex andT_(m)-15° C. for RNA-DNA duplex. Optionally, the degree of dissociationmay be assessed after each wash step to determine the need forsubsequent, higher stringency wash steps.

[0210] High stringency conditions may be used to select for nucleic acidsequences with high degrees of identity to the disclosed sequences. Anexample of stringent hybridization conditions obtained in a filter-basedmethod such as a Southern or northern blot for hybridization ofcomplementary nucleic acids that have more than 100 complementaryresidues is about 5° C. to 20° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Conditions used for hybridization may include about 0.02 M to about 0.15M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS orabout 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodiumcitrate, at hybridization temperatures between about 50° C. and about70° C. More preferably, high stringency conditions are about 0.02 Msodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 Msodium citrate, at a temperature of about 50° C. Nucleic acid moleculesthat hybridize under stringent conditions will typically hybridize to aprobe based on either the entire DNA molecule or selected portions, forexample, to a unique subsequence, of the DNA.

[0211] Stringent salt concentration will ordinarily be less than about750 mM NaCl and 75 mM trisodium citrate. Increasingly stringentconditions may be obtained with less than about 500 mM NaCl and 50 mMtrisodium citrate, to even greater stringency with less than about 250mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can beobtained in the absence of organic solvent, for example, formamide,whereas high stringency hybridization may be obtained in the presence ofat least about 35% formamide, and more preferably at least about 50%formamide. Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. with formamidepresent. Varying additional parameters, such as hybridization time, theconcentration of detergent, for example, sodium dodecyl sulfate (SDS)and ionic strength, are well known to those skilled in the art. Variouslevels of stringency are accomplished by combining these variousconditions as needed.

[0212] The washing steps that follow hybridization may also vary instringency; the post-hybridization wash steps primarily determinehybridization specificity, with the most critical factors beingtemperature and the ionic strength of the final wash solution. Washstringency can be increased by decreasing salt concentration or byincreasing temperature. Stringent salt concentration for the wash stepswill preferably be less than about 30 mM NaCl and 3 mM trisodiumcitrate, and most preferably less than about 15 mM NaCl and 1.5 mMtrisodium citrate.

[0213] Thus, hybridization and wash conditions that may be used to bindand remove polynucleotides with less than the desired homology to thenucleic acid sequences or their complements that encode the presenttranscription factors include, for example:

[0214] 6×SSC at 65° C.;

[0215] 50% formamide, 4×SSC at 42° C.; or

[0216] 0.5×SSC, 0.1% SDS at 65° C.;

[0217] with, for example, two wash steps of 10-30 minutes each.. Usefulvariations on these conditions will be readily apparent to those skilledin the art.

[0218] A person of skill in the art would not expect substantialvariation among polynucleotide species encompassed within the scope ofthe present invention because the highly stringent conditions set forthin the above formulae yield structurally similar polynucleotides.

[0219] If desired, one may employ wash steps of even greater stringency,including about 0.2×SSC, 0.1% SDS at 65° C. and washing twice, each washstep being about 30 min, or about 0.1×SSC, 0.1% SDS at 65° C. andwashing twice for 30 min. The temperature for the wash solutions willordinarily be at least about 25° C., and for greater stringency at leastabout 42° C. Hybridization stringency may be increased further by usingthe same conditions as in the hybridization steps, with the washtemperature raised about 3° C. to about 5° C., and stringency may beincreased even further by using the same conditions except the washtemperature is raised about 6° C. to about 9° C. For identification ofless closely related homolog, wash steps may be performed at a lowertemperature, for example, 50° C.

[0220] An example of a low stringency wash step employs a solution andconditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and0.1% SDS over 30 min. Greater stringency may be obtained at 42° C. in 15mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Evenhigher stringency wash conditions are obtained at 65° C.-68° C. in asolution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Washprocedures will generally employ at least two final wash steps.Additional variations on these conditions will be readily apparent tothose skilled in the art (see, for example, U.S. patent application No.20010010913).

[0221] Stringency conditions can be selected such that anoligonucleotide that is perfectly complementary to the codingoligonucleotide hybridizes to the coding oligonucleotide with at leastabout a 5-10× higher signal to noise ratio than the ratio forhybridization of the perfectly complementary oligonucleotide to anucleic acid encoding a transcription factor known as of the filing dateof the application. It may be desirable to select conditions for aparticular assay such that a higher signal to noise ratio, that is,about 15× or more, is obtained. Accordingly, a subject nucleic acid willhybridize to a unique coding oligonucleotide with at least a 2× orgreater signal to noise ratio as compared to hybridization of the codingoligonucleotide to a nucleic acid encoding known polypeptide. Theparticular signal will depend on the label used in the relevant assay,for example, a fluorescent label, a colorimetric label, a radioactivelabel, or the like. Labeled hybridization or PCR probes for detectingrelated polynucleotide sequences may be produced by oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide.

[0222] Identifying Polynucleotides or Nucleic Acids with ExpressionLibraries

[0223] In addition to hybridization methods, transcription factorhomolog polypeptides can be obtained by screening an expression libraryusing antibodies specific for one or more transcription factors. Withthe provision herein of the disclosed transcription factor, andtranscription factor homolog nucleic acid sequences, the encodedpolypeptide(s) can be expressed and purified in a heterologousexpression system (for example, E. coli) and used to raise antibodies(monoclonal or polyclonal) specific for the polypeptide(s) in question.Antibodies can also be raised against synthetic peptides derived fromtranscription factor, or transcription factor homolog, amino acidsequences. Methods of raising antibodies are well known in the art andare described in Harlow and Lane (1988), Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. Such antibodies canthen be used to screen an expression library produced from the plantfrom which it is desired to clone additional transcription factorhomologs, using the methods described above. The selected cDNAs can beconfirmed by sequencing and enzymatic activity.

[0224] Sequence Variations

[0225] Due to the degeneracy of the genetic code, many differentpolynucleotides can encode identical and/or substantially similarpolypeptides in addition to those sequences illustrated in the SequenceListing. A “variant” of a transcription factor may have an amino acidsequence that is different by one or more deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent transcription factor. Thus, it willbe readily appreciated by those of skill in the art, that any of avariety of polynucleotide sequences is capable of encoding thetranscription factors and transcription factor homolog polypeptides ofthe invention. Variant nucleic acids having a sequence that differs fromthe sequences shown in the Sequence Listing, or complementary sequences,that encode functionally equivalent peptides (i.e., peptides having somedegree of equivalent or similar biological activity) but differ insequence from the sequence shown in the sequence listing due todegeneracy in the genetic code, are also within the scope of theinvention.

[0226] The polypeptide variant may have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties.Deliberate amino acid substitutions may thus be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the functional or biological activity of the transcription factor isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine (for more detail onconservative substitutions, see Table 2). More rarely, a variant mayhave “non-conservative” changes, for example, replacement of a glycinewith a tryptophan. Similar minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing functional or biological activity may be found using computerprograms well known in the art, for example, DNASTAR software (see U.S.Pat. No. 5,840,544).

[0227] Altered or variant polynucleotide sequences encoding polypeptidesinclude those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide encoding apolypeptide with at least one functional characteristic of the instantpolypeptides. Included within this definition are polymorphisms whichmay or may not be readily detectable using a particular oligonucleotideprobe of the polynucleotide encoding the instant polypeptides, andimproper or unexpected hybridization to allelic variants, with a locusother than the normal chromosomal locus for the polynucleotide sequenceencoding the instant polypeptides.

[0228] Allelic variant refers to any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (i.e., no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene. Splice variant refersto alternative forms of RNA transcribed from a gene. Splice variationarises naturally through use of alternative splicing sites within atranscribed RNA molecule, or less commonly between separatelytranscribed RNA molecules, and may result in several mRNAs transcribedfrom the same gene. Splice variants may encode polypeptides havingaltered amino acid sequence. The term splice variant is also used hereinto denote a protein encoded by a splice variant of an mRNA transcribedfrom a gene.

[0229] Those skilled in the art would recognize that SEQ ID NO: 2, forexample, represents a single transcription factor; allelic variation andalternative splicing may be expected to occur. Allelic variants of SEQID NO: 1 can be cloned by probing cDNA or genomic libraries fromdifferent individual organisms according to standard procedures. Allelicvariants of the DNA sequence shown in SEQ ID NO: 1, including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention, asare proteins which are allelic variants of SEQ ID NO: 2. cDNAs generatedfrom alternatively spliced mRNAs, which retain the properties of thetranscription factor are included within the scope of the presentinvention, as are polypeptides encoded by such cDNAs and mRNAs. Allelicvariants and splice variants of these sequences can be cloned by probingcDNA or genomic libraries from different individual organisms or tissuesaccording to standard procedures known in the art (see U.S. Pat. No.6,388,064).

[0230] For example, Table 1 illustrates, for example, that the codonsAGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine.Accordingly, at each position in the sequence where there is a codonencoding serine, any of the above trinucleotide sequences can be usedwithout altering the encoded polypeptide. TABLE 1 Amino acid PossibleCodons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C TGC TGT Asparticacid Asp D GAC GAT Glutamic acid Glu E GAA GAG Phenylalanine Phe F TTCTTT Glycine Gly G GGA GGC GGG GGT Histidine His H CAC CAT Isoleucine IleI ATA ATC ATT Lysine Lys K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTTMethionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC CCGCCT Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGTSerine Ser S AGC AGT TCA TCC TCG TCT Threonine Thr T ACA ACC ACG ACTValine Val V GTA GTC GTG GTT Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

[0231] Sequence alterations that do not change the amino acid sequenceencoded by the polynucleotide are termed “silent” variations. With theexception of the codons ATG and TGG, encoding methionine and tryptophan,respectively, any of the possible codons for the same amino acid can besubstituted by a variety of techniques, for example, site-directedmutagenesis, available in the art. Accordingly, any and all suchvariations of a sequence selected from the above table are a feature ofthe invention.

[0232] In addition to silent variations, other conservative variationsthat alter one, or a few amino acids in the encoded polypeptide, can bemade without altering the function of the polypeptide, theseconservative variants are, likewise, a feature of the invention.

[0233] For example, substitutions, deletions and insertions introducedinto the sequences provided in the Sequence Listing are also envisionedby the invention. Such sequence modifications can be engineered into asequence by site-directed mutagenesis (Wu (editor) Meth. Enzymol. (1993)vol. 217, Academic Press) or the other methods noted below. Amino acidsubstitutions are typically of single residues; insertions usually willbe on the order of about from 1 to 10 amino acid residues; and deletionswill range about from 1 to 30 residues. In preferred embodiments,deletions or insertions are made in adjacent pairs, for example, adeletion of two residues or insertion of two residues. Substitutions,deletions, insertions or any combination thereof can be combined toarrive at a sequence. The mutations that are made in the polynucleotideencoding the transcription factor should not place the sequence out ofreading frame and should not create complementary regions that couldproduce secondary mRNA structure. Preferably, the polypeptide encoded bythe DNA performs the desired function.

[0234] Conservative substitutions are those in which at least oneresidue in the amino acid sequence has been removed and a differentresidue inserted in its place. Such substitutions generally are made inaccordance with the Table 2 when it is desired to maintain the activityof the protein. Table 2 shows amino acids which can be substituted foran amino acid in a protein and which are typically regarded asconservative substitutions. TABLE 2 Residue Conservative SubstitutionsAla Ser Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro HisAsn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met;Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0235] Similar substitutions are those in which at least one residue inthe amino acid sequence has been removed and a different residueinserted in its place. Such substitutions generally are made inaccordance with the Table 3 when it is desired to maintain the activityof the protein. Table 3 shows amino acids which can be substituted foran amino acid in a protein and which are typically regarded asstructural and functional substitutions. For example, a residue incolumn 1 of Table 3 may be substituted with residue in column 2; inaddition, a residue in column 2 of Table 3 may be substituted with theresidue of column 1. TABLE 3 Residue Similar Substitutions Ala Ser; Thr;Gly; Val; Leu; Ile Arg Lys; His; Gly Asn Gln; His; Gly; Ser; Thr AspGlu, Ser; Thr Gln Asn; Ala Cys Ser; Gly Glu Asp Gly Pro; Arg His Asn;Gln; Tyr; Phe; Lys; Arg Ile Ala; Leu; Val; Gly; Met Leu Ala; Ile; Val;Gly; Met Lys Arg; His; Gln; Gly; Pro Met Leu; Ile; Phe Phe Met; Leu;Tyr; Trp; His; Val; Ala Ser Thr; Giy; Asp; Ala; Val; Ile; His Thr Ser;Val; Ala; Gly Trp Tyr; Phe; His Tyr Trp; Phe; His Val Ala; Ile; Leu;Gly; Thr; Ser; Glu

[0236] Substitutions that are less conservative than those in Table 2can be selected by picking residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in proteinproperties will be those in which (a) a hydrophilic residue, forexample, seryl or threonyl, is substituted for (or by) a hydrophobicresidue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl;(b) a cysteine or proline is substituted for (or by) any other residue;(c) a residue having an electropositive side chain, for example, lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, for example, glutamyl or aspartyl; or (d) a residue having abulky side chain, for example, phenylalanine, is substituted for (or by)one not having a side chain, for example, glycine.

[0237] Further Modifying Sequences of the Invention—Mutation/ForcedEvolution

[0238] In addition to generating silent or conservative substitutions asnoted, above, the present invention optionally includes methods ofmodifying the sequences of the Sequence Listing. In the methods, nucleicacid or protein modification methods are used to alter the givensequences to produce new sequences and/or to chemically or enzymaticallymodify given sequences to change the properties of the nucleic acids orproteins.

[0239] Thus, in one embodiment, given nucleic acid sequences aremodified, for example, according to standard mutagenesis or artificialevolution methods to produce modified sequences. The modified sequencesmay be created using purified natural polynucleotides isolated from anyorganism or may be synthesized from purified compositions and chemicalsusing chemical means well know to those of skill in the art. Forexample, Ausubel, supra, provides additional details on mutagenesismethods. Artificial forced evolution methods are described, for example,by Stemmer (1994) Nature 370: 389-391, Stemmer (1994) Proc. Natl. Acad.Sci. 91: 10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and6,242,568. Methods for engineering synthetic transcription factors andother polypeptides are described, for example, by Zhang et al. (2000) J.Biol. Chem. 275: 33850-33860, Liu et al. (2001) J. Biol. Chem. 276:11323-11334, and Isalan et al. (2001) Nature Biotechnol. 19: 656-660.Many other mutation and evolution methods are also available andexpected to be within the skill of the practitioner.

[0240] Similarly, chemical or enzymatic alteration of expressed nucleicacids and polypeptides can be performed by standard methods. Forexample, sequence can be modified by addition of lipids, sugars,peptides, organic or inorganic compounds, by the inclusion of modifiednucleotides or amino acids, or the like. For example, proteinmodification techniques are illustrated in Ausubel, supra. Furtherdetails on chemical and enzymatic modifications can be found herein.These modification methods can be used to modify any given sequence, orto modify any sequence produced by the various mutation and artificialevolution modification methods noted herein.

[0241] Accordingly, the invention provides for modification of any givennucleic acid by mutation, evolution, chemical or enzymatic modification,or other available methods, as well as for the products produced bypracticing such methods, for example, using the sequences herein as astarting substrate for the various modification approaches.

[0242] For example, optimized coding sequence containing codonspreferred by a particular prokaryotic or eukaryotic host can be used forexample, to increase the rate of translation or to produce recombinantRNA transcripts having desirable properties, such as a longer half-life,as compared with transcripts produced using a non-optimized sequence.Translation stop codons can also be modified to reflect host preference.For example, preferred stop codons for Saccharomyces cerevisiae andmammals are TAA and TGA, respectively. The preferred stop codon formonocotyledonous plants is TGA, whereas insects and E. coli prefer touse TAA as the stop codon.

[0243] The polynucleotide sequences of the present invention can also beengineered in order to alter a coding sequence for a variety of reasons,including but not limited to, alterations which modify the sequence tofacilitate cloning, processing and/or expression of the gene product.For example, alterations are optionally introduced using techniqueswhich are well known in the art, for example, site-directed mutagenesis,to insert new restriction sites, to alter glycosylation patterns, tochange codon preference, to introduce splice sites, etc.

[0244] Furthermore, a fragment or domain derived from any of thepolypeptides of the invention can be combined with domains derived fromother transcription factors or synthetic domains to modify thebiological activity of a transcription factor. For instance, aDNA-binding domain derived from a transcription factor of the inventioncan be combined with the activation domain of another transcriptionfactor or with a synthetic activation domain. A transcription activationdomain assists in initiating transcription from a DNA-binding site.Examples include the transcription activation region of VP16 or GAL4(Moore et al. (1998) Proc. Natl. Acad. Sci. 95: 376-381; and Aoyama etal. (1995) Plant Cell 7: 1773-1785), peptides derived from bacterialsequences (Ma and Ptashne (1987) Cell 51: 113-119) and syntheticpeptides (Giniger and Ptashne (1987) Nature 330: 670-672).

[0245] Expression and Modification of Polypeptides

[0246] Typically, polynucleotide sequences of the invention areincorporated into recombinant DNA (or RNA) molecules that directexpression of polypeptides of the invention in appropriate host cells,transgenic plants, in vitro translation systems, or the like. Due to theinherent degeneracy of the genetic code, nucleic acid sequences whichencode substantially the same or a functionally equivalent amino acidsequence can be substituted for any listed sequence to provide forcloning and expressing the relevant homolog.

[0247] Vectors, Promoters, and Expression Systems

[0248] The present invention includes recombinant constructs comprisingone or more of the nucleic acid sequences herein. The constructstypically comprise a vector, such as a plasmid, a cosmid, a phage, avirus (for example, a plant virus), a bacterial artificial chromosome(BAC), a yeast artificial chromosome (YAC), or the like, into which anucleic acid sequence of the invention has been inserted, in a forwardor reverse orientation. In a preferred aspect of this embodiment, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and are commercially available.

[0249] General texts that describe molecular biological techniquesuseful herein, including the use and production of vectors, promotersand many other relevant topics, include Berger, Sambrook and Ausubel,supra. Any of the identified sequences can be incorporated into acassette or vector, for example, for expression in plants. A number ofexpression vectors suitable for stable transformation of plant cells orfor the establishment of transgenic plants have been described includingthose described in Weissbach and Weissbach (1989) Methods for PlantMolecular Biology, Academic Press, and Gelvin et al. (1990) PlantMolecular Biology Manual, Kluwer Academic Publishers. Specific examplesinclude those derived from a Ti plasmid of Agrobacterium tumefaciens, aswell as those disclosed by Herrera-Estrella et al. (1983) Nature 303:209, Bevan (1984) Nucl Acid Res. 12: 8711-8721, Klee (1985)Bio/Technology 3: 637-642, for dicotyledonous plants.

[0250] Alternatively, non-Ti vectors can be used to transfer the DNAinto monocotyledonous plants and cells by using free DNA deliverytechniques. Such methods can involve, for example, the use of liposomes,electroporation, microprojectile bombardment, silicon carbide whiskers,and viruses. By using these methods transgenic plants such as wheat,rice (Christou (1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm(1990) Plant Cell 2: 603-618) can be produced. An immature embryo canalso be a good target tissue for monocots for direct DNA deliverytechniques by using the particle gun (Weeks et al. (1993) Plant Physiol.102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux(1994) Plant Physiol. 104: 37-48, and for Agrobacterium-mediated DNAtransfer (Ishida et al. (1996) Nature Biotechnol. 14: 745-750).

[0251] Typically, plant transformation vectors include one or morecloned plant coding sequence (genomic or cDNA) under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant transformation vectors typically also contain apromoter (for example, a regulatory region controlling inducible orconstitutive, environmentally-or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, anRNA processing signal (such as intron splice sites), a transcriptiontermination site, and/or a polyadenylation signal.

[0252] Examples of constitutive plant promoters which can be useful forexpressing the TF sequence include: the cauliflower mosaic virus (CaMV)35S promoter, which confers constitutive, high-level expression in mostplant tissues (see, for example, Odell et al. (1985) Nature 313:810-812); the nopaline synthase promoter (An et al. (1988) PlantPhysiol. 88: 547-552); and the octopine synthase promoter (Fromm et al.(1989) Plant Cell 1: 977-984).

[0253] A variety of plant gene promoters that regulate gene expressionin response to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner can be used for expression of a TFsequence in plants. Choice of a promoter is based largely on thephenotype of interest and is determined by such factors as tissue (forexample, seed, fruit, root, pollen, vascular tissue, flower, carpel,etc.), inducibility (for example, in response to wounding, heat, cold,drought, light, pathogens, etc.), timing, developmental stage, and thelike. Numerous known promoters have been characterized and can favorablybe employed to promote expression of a polynucleotide of the inventionin a transgenic plant or cell of interest. For example, tissue specificpromoters include: seed-specific promoters (such as the napin, phaseolinor DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specificpromoters that are active during fruit ripening (such as the dru 1promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No.4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988)Plant Mol. Biol. 11: 651-662), root-specific promoters, such as thosedisclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186,pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No.5,792,929), promoters active in vascular tissue (Ringli et al. (1998)Plant Mol. Biol. 37: 977-988), flower-specific (Kaiser et al, (1995)Plant Mol. Biol. 28: 231-243), pollen (Baerson et al. (1994) Plant Mol.Biol. 26: 1947-1959), carpels (Ohl et al. (1990) Plant Cell 2: 837-848),pollen and ovules (Baerson et al. (1993) Plant Mol. Biol. 22: 255-267),auxin-inducible promoters (such as that described in van der Kop et al.(1999) Plant Mol. Biol. 39: 979-990 or Baumann et al. (1999) Plant Cell11: 323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) PlantMol. Biol. 38: 743-753), promoters responsive to gibberellin (Shi et al.(1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) 38:817-825) and the like. Additional promoters are those that elicitexpression in response to heat (Ainley et al. (1993) Plant Mol. Biol.22: 13-23), light (for example, the pea rbcS-3A promoter, Kuhlemeier etal. (1989) Plant Cell 1: 471-478, and the maize rbcS promoter, Schaffnerand Sheen (1991) Plant Cell 3: 997-1012); wounding (for example, wunI,Siebertz et al. (1989) Plant Cell 1: 961-968); pathogens (such as thePR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners et al. (1998)Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonateor salicylic acid (Gatz et al. (1997) Ann. Rev. Plant Physiol. PlantMol. Biol. 48: 89-108). In addition, the timing of the expression can becontrolled by using promoters such as those acting at senescence (Gan etal. (1995) Science 270: 1986-1988); or late seed development (Odell etal. (1994) Plant Physiol. 106: 447-458).

[0254] Plant expression vectors can also include RNA processing signalsthat can be positioned within, upstream or downstream of the codingsequence. In addition, the expression vectors can include additionalregulatory sequences from the 3′-untranslated region of plant genes, forexample, a 3′ terminator region to increase mRNA stability of the mRNA,such as the PI-II terminator region of potato or the octopine ornopaline synthase 3′ terminator regions.

[0255] Additional Expression Elements

[0256] Specific initiation signals can aid in efficient translation ofcoding sequences. These signals can include, for example, the ATGinitiation codon and adjacent sequences. In cases where a codingsequence, its initiation codon and upstream sequences are inserted intothe appropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only coding sequence (forexample, a mature protein coding sequence), or a portion thereof, isinserted, exogenous transcriptional control signals including the ATGinitiation codon can be separately provided. The initiation codon isprovided in the correct reading frame to facilitate transcription.Exogenous transcriptional elements and initiation codons can be ofvarious origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers appropriate tothe cell system in use.

[0257] Expression Hosts

[0258] The present invention also relates to host cells which aretransduced with vectors of the invention, and the production ofpolypeptides of the invention (including fragments thereof) byrecombinant techniques. Host cells are genetically engineered (i.e.,nucleic acids are introduced, for example, transduced, transformed ortransfected) with the vectors of this invention, which may be, forexample, a cloning vector or an expression vector comprising therelevant nucleic acids herein. The vector is optionally a plasmid, aviral particle, a phage, a naked nucleic acid, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants, oramplifying the relevant gene. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to those skilled inthe art and in the references cited herein, including, Sambrook andAusubel.

[0259] The host cell can be a eukaryotic cell, such as a yeast cell, ora plant cell, or the host cell can be a prokaryotic cell, such as abacterial cell. Plant protoplasts are also suitable for someapplications. For example, the DNA fragments are introduced into planttissues, cultured plant cells or plant protoplasts by standard methodsincluding electroporation (Fromm et al. (1985) Proc. Natl. Acad. Sci.82: 5824, infection by viral vectors such as cauliflower mosaic virus(CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors, (AcademicPress, New York) pp. 549-560; U.S. Pat. No. 4,407,956), high velocityballistic penetration by small particles with the nucleic acid eitherwithin the matrix of small beads or particles, or on the surface (Kleinet al. (1987) Nature 327: 70-73), use of pollen as vector (WO 85/01856),or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNAplasmid in which DNA fragments are cloned. The T-DNA plasmid istransmitted to plant cells upon infection by Agrobacterium tumefaciens,and a portion is stably integrated into the plant genome (Horsch et al.(1984) Science 233: 496-498; Fraley et al. (1983) Proc. Natl. Acad. Sci.80: 4803).

[0260] The cell can include a nucleic acid of the invention that encodesa polypeptide, wherein the cell expresses a polypeptide of theinvention. The cell can also include vector sequences, or the like.Furthermore, cells and transgenic plants that include any polypeptide ornucleic acid above or throughout this specification, for example,produced by transduction of a vector of the invention, are an additionalfeature of the invention.

[0261] Modified Amino Acid Residues

[0262] Polypeptides of the invention may contain one or more modifiedamino acid residues. The presence of modified amino acids may beadvantageous in, for example, increasing polypeptide half-life, reducingpolypeptide antigenicity or toxicity, increasing polypeptide storagestability, or the like. Amino acid residue(s) are modified, for example,co-translationally or post-translationally during recombinant productionor modified by synthetic or chemical means.

[0263] Non-limiting examples of a modified amino acid residue includeincorporation or other use of acetylated amino acids, glycosylated aminoacids, sulfated amino acids, prenylated (for example, farnesylated,geranylgeranylated) amino acids, PEG modified (for example, “PEGylated”)amino acids, biotinylated amino acids, carboxylated amino acids,phosphorylated amino acids, etc. References adequate to guide one ofskill in the modification of amino acid residues are replete throughoutthe literature.

[0264] The modified amino acid residues may prevent or increase affinityof the polypeptide for another molecule, including, but not limited to,polynucleotide, proteins, carbohydrates, lipids and lipid derivatives,and other organic or synthetic compounds.

[0265] Identification of Additional Factors

[0266] A transcription factor provided by the present invention can alsobe used to identify additional endogenous or exogenous molecules thatcan affect a phentoype or trait of interest. On the one hand, suchmolecules include organic (small or large molecules) and/or inorganiccompounds that affect expression of (i.e., regulate) a particulartranscription factor. Alternatively, such molecules include endogenousmolecules that are acted upon either at a transcriptional level by atranscription factor of the invention to modify a phenotype as desired.For example, the transcription factors can be employed to identify oneor more downstream gene with which is subject to a regulatory effect ofthe transcription factor. In one approach, a transcription factor ortranscription factor homolog of the invention is expressed in a hostcell, for example, a transgenic plant cell, tissue or explant, andexpression products, either RNA or protein, of likely or random targetsare monitored, for example, by hybridization to a microarray of nucleicacid probes corresponding to genes expressed in a tissue or cell type ofinterest, by two-dimensional gel electrophoresis of protein products, orby any other method known in the art for assessing expression of geneproducts at the level of RNA or protein. Alternatively, a transcriptionfactor of the invention can be used to identify promoter sequences(i.e., binding sites) involved in the regulation of a downstream target.After identifying a promoter sequence, interactions between thetranscription factor and the promoter sequence can be modified bychanging specific nucleotides in the promoter sequence or specific aminoacids in the transcription factor that interact with the promotersequence to alter a plant trait. Typically, transcription factorDNA-binding sites are identified by gel shift assays. After identifyingthe promoter regions, the promoter region sequences can be employed indouble-stranded DNA arrays to identify molecules that affect theinteractions of the transcription factors with their promoters (Bulyk etal. (1999) Nature Biotechnol. 17: 573-577).

[0267] The identified transcription factors are also useful to identifyproteins that modify the activity of the transcription factor. Suchmodification can occur by covalent modification, such as byphosphorylation, or by protein-protein (homo- or heteropolymer)interactions. Any method suitable for detecting protein-proteininteractions can be employed. Among the methods that can be employed areco-immunoprecipitation, cross-linking and co-purification throughgradients or chromatographic columns, and the two-hybrid yeast system.

[0268] The two-hybrid system detects protein interactions in vivo and isdescribed in Chien et al. ((1991), Proc. Natl. Acad. Sci. 88: 9578-9582)and is commercially available from Clontech (Palo Alto, Calif.). In sucha system, plasmids are constructed that encode two hybrid proteins: oneconsists of the DNA-binding domain of a transcription activator proteinfused to the TF polypeptide and the other consists of the transcriptionactivator protein's activation domain fused to an unknown protein thatis encoded by a cDNA that has been recombined into the plasmid as partof a cDNA library. The DNA-binding domain fusion plasmid and the cDNAlibrary are transformed into a strain of the yeast Saccharomycescerevisiae that contains a reporter gene (for example, lacZ) whoseregulatory region contains the transcription activator's binding site.Either hybrid protein alone cannot activate transcription of thereporter gene. Interaction of the two hybrid proteins reconstitutes thefunctional activator protein and results in expression of the reportergene, which is detected by an assay for the reporter gene product. Then,the library plasmids responsible for reporter gene expression areisolated and sequenced to identify the proteins encoded by the libraryplasmids. After identifying proteins that interact with thetranscription factors, assays for compounds that interfere with the TFprotein-protein interactions can be preformed.

[0269] Identification of Modulators

[0270] In addition to the intracellular molecules described above,extracellular molecules that alter activity or expression of atranscription factor, either directly or indirectly, can be identified.For example, the methods can entail first placing a candidate moleculein contact with a plant or plant cell. The molecule can be introduced bytopical administration, such as spraying or soaking of a plant, and thenthe molecule's effect on the expression or activity of the TFpolypeptide or the expression of the polynucleotide monitored. Changesin the expression of the TF polypeptide can be monitored by use ofpolyclonal or monoclonal antibodies, gel electrophoresis or the like.Changes in the expression of the corresponding polynucleotide sequencecan be detected by use of microarrays, Northerns, quantitative PCR, orany other technique for monitoring changes in mRNA expression. Thesetechniques are exemplified in Ausubel et al., editors, Current Protocolsin Molecular Biology, John Wiley & Sons (1998, and supplements through2001). Such changes in the expression levels can be correlated withmodified plant traits and thus identified molecules can be useful forsoaking or spraying on fruit, vegetable and grain crops to modify traitsin plants.

[0271] Essentially any available composition can be tested formodulatory activity of expression or activity of any nucleic acid orpolypeptide herein. Thus, available libraries of compounds such aschemicals, polypeptides, nucleic acids and the like can be tested formodulatory activity. Often, potential modulator compounds can bedissolved in aqueous or organic (for example, DMSO-based) solutions foreasy delivery to the cell or plant of interest in which the activity ofthe modulator is to be tested. Optionally, the assays are designed toscreen large modulator composition libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (for example, in microtiter formatson microtiter plates in robotic assays).

[0272] In one embodiment, high throughput screening methods involveproviding a combinatorial library containing a large number of potentialcompounds (potential modulator compounds). Such “combinatorial chemicallibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as target compounds.

[0273] A combinatorial chemical library can be, for example, acollection of diverse chemical compounds generated by chemical synthesisor biological synthesis. For example, a combinatorial chemical librarysuch as a polypeptide library is formed by combining a set of chemicalbuilding blocks (for example, in one example, amino acids) in everypossible way for a given compound length (i.e., the number of aminoacids in a polypeptide compound of a set length). Exemplary librariesinclude peptide libraries, nucleic acid libraries, antibody libraries(see, for example, Vaughn et al. (1996) Nature Biotechnol. 14: 309-314and PCT/US96/10287), carbohydrate libraries (see, for example, Liang etal. Science (1996) 274: 1520-1522 and U.S. Pat. No. 5,593,853), peptidenucleic acid libraries (see, for example, U.S. Pat. No. 5,539,083), andsmall organic molecule libraries (see, for example, benzodiazepines,Baum (1993) Chem Eng. News January 18, p. 33; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337) and the like.

[0274] Preparation and screening of combinatorial or other libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, forexample, U.S. Pat. No. 5,010,175; Furka, (1991) Int. J. Pept. Prot. Res.37: 487-493; and Houghton et al. (1991) Nature 354: 84-88). Otherchemistries for generating chemical diversity libraries can also beused.

[0275] In addition, as noted, compound screening equipment forhigh-throughput screening is generally available, for example, using anyof a number of well known robotic systems that have also been developedfor solution phase chemistries useful in assay systems. These systemsinclude automated workstations including an automated synthesisapparatus and robotic systems utilizing robotic arms. Any of the abovedevices are suitable for use with the present invention, for example,for high-throughput screening of potential modulators. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art.

[0276] Indeed, entire high-throughput screening systems are commerciallyavailable. These systems typically automate entire procedures includingall sample and reagent pipetting, liquid dispensing, timed incubations,and final readings of the microplate in detector(s) appropriate for theassay. These configurable systems provide high throughput and rapidstart up as well as a high degree of flexibility and customization.Similarly, microfluidic implementations of screening are alsocommercially available.

[0277] The manufacturers of such systems provide detailed protocols thevarious high throughput. Thus, for example, Zymark Corp. providestechnical bulletins describing screening systems for detecting themodulation of gene transcription, ligand binding, and the like. Theintegrated systems herein, in addition to providing for sequencealignment and, optionally, synthesis of relevant nucleic acids, caninclude such screening apparatus to identify modulators that have aneffect on one or more polynucleotides or polypeptides according to thepresent invention.

[0278] In some assays it is desirable to have positive controls toensure that the components of the assays are working properly. At leasttwo types of positive controls are appropriate. That is, knowntranscriptional activators or inhibitors can be incubated withcells/plants/etc. in one sample of the assay, and the resultingincrease/decrease in transcription can be detected by measuring theresulting increase in RNA/protein expression, etc., according to themethods herein. It will be appreciated that modulators can also becombined with transcriptional activators or inhibitors to findmodulators that inhibit transcriptional activation or transcriptionalrepression. Either expression of the nucleic acids and proteins hereinor any additional nucleic acids or proteins activated by the nucleicacids or proteins herein, or both, can be monitored.

[0279] In an embodiment, the invention provides a method for identifyingcompositions that modulate the activity or expression of apolynucleotide or polypeptide of the invention. For example, a testcompound, whether a small or large molecule, is placed in contact with acell, plant (or plant tissue or explant), or composition comprising thepolynucleotide or polypeptide of interest and a resulting effect on thecell, plant, (or tissue or explant) or composition is evaluated bymonitoring, either directly or indirectly, one or more of: expressionlevel of the polynucleotide or polypeptide, activity (or modulation ofthe activity) of the polynucleotide or polypeptide. In some cases, analteration in a plant phenotype can be detected following contact of aplant (or plant cell, or tissue or explant) with the putative modulator,for example, by modulation of expression or activity of a polynucleotideor polypeptide of the invention. Modulation of expression or activity ofa polynucleotide or polypeptide of the invention may also be caused bymolecular elements in a signal transduction second messenger pathway andsuch modulation can affect similar elements in the same or anothersignal transduction second messenger pathway.

[0280] Subsequences

[0281] Also contemplated are uses of polynucleotides, also referred toherein as oligonucleotides, typically having at least 12 bases,preferably at least 15, more preferably at least 20, 30, or 50 bases,which hybridize under at least highly stringent (or ultra-high stringentor ultra-ultra-high stringent conditions) conditions to a polynucleotidesequence described above. The polynucleotides may be used as probes,primers, sense and antisense agents, and the like, according to methodsas noted supra.

[0282] Subsequences of the polynucleotides of the invention, includingpolynucleotide fragments and oligonucleotides are useful as nucleic acidprobes and primers. An oligonucleotide suitable for use as a probe orprimer is at least about 15 nucleotides in length, more often at leastabout 18 nucleotides, often at least about 21 nucleotides, frequently atleast about 30 nucleotides, or about 40 nucleotides, or more in length.A nucleic acid probe is useful in hybridization protocols, for example,to identify additional polypeptide homologs of the invention, includingprotocols for microarray experiments. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, for example, bythe polymerase chain reaction (PCR) or other nucleic-acid amplificationmethods. See Sambrook and Ausubel, supra.

[0283] In addition, the invention includes an isolated or recombinantpolypeptide including a subsequence of at least about 15 contiguousamino acids encoded by the recombinant or isolated polynucleotides ofthe invention. For example, such polypeptides, or domains or fragmentsthereof, can be used as immunogens, for example, to produce antibodiesspecific for the polypeptide sequence, or as probes for detecting asequence of interest. A subsequence can range in size from about 15amino acids in length up to and including the full length of thepolypeptide.

[0284] To be encompassed by the present invention, an expressedpolypeptide which comprises such a polypeptide subsequence performs atleast one biological function of the intact polypeptide in substantiallythe same manner, or to a similar extent, as does the intact polypeptide.For example, a polypeptide fragment can comprise a recognizablestructural motif or functional domain such as a DNA binding domain thatbinds to a specific DNA promoter region, an activation domain or adomain for protein-protein interactions.

[0285] 2. Description of the Invention

[0286] The present invention relates to a method for modifying thebiomass of a plant. By modifying a plant's biomass, any of a number ofdesirable plant traits can be modified, including plant yield, largerroot mass to increase the tolerance to certain environmental stresses,modified leaf area in response to different light intensities, increasedleaf mass for human or animal consumption, or the like.

[0287] The present invention also relates to a method for increasing thelevels of a cell protectant in a cell. By increasing the levels of thecell protectant, a cell's or plant's response to a variety ofenvironmental stresses can be improved. The type of environmental stressthat can be modified includes the cold or freezing tolerance of the cellor the drought or salinity tolerance of the cell or plant. The cellprotectant may be a cryoprotectant, an osmoprotectant or the like.Exemplary cell protectants include proline, sugars, lipids or the like.The method can also be used to increase levels of a number of cellprotectants simultaneously.

[0288] The method entails altering the levels of a polynucleotideencoding a CBF-related polypeptide in a transformed plant. Thetransformed plant may be generated by transforming a plant with anexpression vector comprising a polynucleotide sequence encoding aCBF-related polypeptide or by breeding after the initial transformationof a parental plant comprising the expression vector. Transformed plantsare then selected that express the polynucleotide. The resulting plantsare plants with increased biomass, including, for example, larger leavesand/or larger root systems, or plants that produce higher levels of cellprotectants.

[0289] The method also entails generating a transformed cell or plantthat overexpresses a recombinant polynucleotide encoding a CBF-relatedpolypeptide. The transformed cell or plant may be generated bytransforming a cell or plant with an expression vector comprising apolynucleotide sequence encoding a cold-regulatable polypeptide or bybreeding after the initial transformation of a parental cell comprisingthe expression vector. Transformed cells or plants are then selected forexpression of the polynucleotide and grown. The resulting cells orplants produce higher levels of cell protectants. Higher levels of cellprotectants can be detected either in the absence of or after exposureto cold temperatures (cold acclimation). However, higher levels of cellprotectants are typically observed in cells or plants that have beencold acclimated compared with levels observed in cells or plants thathave not been cold-acclimated.

[0290] By increasing the levels of a single cell protectant or acombination of cell protectants simultaneously in the cell, the cell'stolerance to environmental stresses can be substantially improved, asmeasured for example by a plant's survival rate after exposure tofreezing temperatures or the growth of a plant's roots after exposure todrought conditions or high salinity.

[0291] The present invention also relates to a method for producing acell protectant from a cell or plant. The method entails generating atransformed cell that overexpresses a recombinant polynucleotideencoding a CBF-related polypeptide. Expression of the recombinantpolynucleotide in the cell or plant derived from the transformed cellcauses metabolic pathways that produce or accumulate certain cellprotectants to be turned on so that higher levels of the cellprotectants are produced. For example, we have observed that the keyproline biosynthetic pathway enzyme, P5CS, is expressed at higher levelswhen the CBF-related polynucleotide is overexpressed. Then the cellprotectant, such as proline, sugars or lipids, can be isolated from theplant using well known isolation and purification methods

[0292] The present invention can be applied to modify the biomass orincrease cell protectant levels in or improve the environmental stresstolerance of a variety of plant cells or plants in particular, cells orplants monocots, dicots and gymnosperms. In particular the invention maybe used for modifying the biomass or environmental stress response ofagriculturally important plant species, including but not limited to,crops such as soybean, wheat, corn, potato, cotton, rice, oilseed rape(including canola), sunflower, alfalfa, sugarcane and turf; or fruitsand vegetables, such as banana, blackberry, blueberry, strawberry, andraspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant,grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers,pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato,watermelon, rosaceous fruits (such as apple, peach, pear, cherry, andplum) and vegetable brassicas (such as broccoli, cabbage, cauliflower,Brussels sprouts, and kohlrabi). Other crops, fruits and vegetableswhose phenotype may be changed include barley, currant, avocado, citrusfruits such as oranges, lemons, grapefruit and tangerines, artichoke,cherries, nuts such as the walnut and peanut, endive, leek, roots, suchas arrowroot, beet, cassava, turnip, radish, yam, sweet potato andbeans. The present invention may also be employed to modify plantbiomass and increase cell protectant levels in woody plants, such pine,poplar and eucalyptus.

[0293] A. CBF-Related Polypeptide

[0294] The CBF-related protein may comprise a whole gene coding sequenceor a fragment or domain of a coding sequence. A “fragment or domain”, asreferred to polypeptides, may be a portion of a polypeptide whichperforms at least one biological function of the intact polypeptide insubstantially the same manner or to a similar extent as does the intactpolypeptide. A fragment may comprise, for example, a DNA binding domainthat binds to a specific DNA promoter region (such as the AP2 domain),an activation domain or a domain for protein-protein interactions.Fragments may vary in size from as few as 6 amino acids to the length ofthe intact polypeptide, but are preferably at least 30 amino acids inlength and more preferably at least 60 amino acids in length. Forexample, one can identify any number of 60 amino acid long fragments(1-60, 5-65, 10-70, 15-75, etc.) along the length of the CBF3polypeptide shown in FIG. 13. In reference to a nucleotide sequence “afragment” refers to any sequence of at least 18 consecutive nucleotides,preferably at least 30 nucleotides, more preferably at least 50, of anyof the sequences provided herein.

[0295] The CBF-related polypeptides encompass naturally occurringsequences. Numerous CBF-related proteins have been previously identifiedand include the genes, CBF1, CBF2, and CBF3 (also known as DREB1b,DREB1c, and DREB1a, respectively), which are located in tandem onchromosome 4 in Arabidopsis (Gilmour et al. (1998) supra; Shinwari etal. (1998) Biochem. Biophys. Res. Commun. 250: 161-170). Additionalexamples of CBF-related polypeptides include those described inStockinger et al. PCT publication WO99/38977, and U.S. patentapplication Ser. No. 09/198,119, entitled “Plant Having AlteredEnvironmental Stress Tolerance”, filed Nov. 23, 1998 and U.S.Provisional Patent Application No. 60/165,860 entitled “Method forModifying the Cold Resistance of Plants”, filed Nov. 16, 1999.

[0296] The CBF-related polypeptides may also encompass non-naturallyoccurring sequences that are derivatives of the naturally-occurring CBFsdescribed above. For example, a non-naturally occurring sequence usingdomains of other transcription factors described above fused in frame,but not necessarily adjacent, with functional domains derived from othersequences or sources. Additionally, the invention includes polypeptidesderived from shuffling regions of transcription factors described aboveby methods described in Minshull and Stemmer, U.S. Pat. No. 5,837,458,entitled “Methods and Compositions for Cellular and MetabolicEngineering” and Stemmer and Crameri, U.S. Pat. No. 5,811,238, entitled“Methods for Generating Polynucleotides having Desired Characteristicsby Iterative Selection and Recombination”.

[0297] Substitutions, deletions and insertions introduced intoCBF-related polypeptides are also envisioned by the invention. Suchsequence modifications can be engineered into a sequence bysite-directed mutagenesis (Wu (editor) Meth. Enzymol. (1993) vol. 217,Academic Press). Amino acid substitutions are typically of singleresidues and may be conservative (such as serine to threonine) ornon-conservative (such as lysine to glutamic acid); insertions usuallywill be on the order of about from 1 to 10 amino acid residues; anddeletions will range about from 1 to 30 residues. In preferredembodiments, deletions or insertions are made in adjacent pairs, forexample, a deletion of two residues or insertion of two residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a sequence.

[0298] Additionally, the CBF-related polypeptide may encompass apolypeptide sequence that is modified by chemical or enzymatic means.The homologous sequence may be a sequence modified by lipids, sugars,peptides, organic or inorganic compounds, by the use of modified aminoacids or the like. Protein modification techniques are illustrated inAusubel et al., editors, (1998) Current Protocols in Molecular Biology,John Wiley & Sons.

[0299] B. Altered Expression of CBF-Related Polypeptide

[0300] Any of the identified sequences may be incorporated into acassette or vector for expression in cells, in particular plant cells. Anumber of expression vectors suitable for stable transformation of plantcells or for the establishment of transgenic plants have been describedincluding those described in Weissbach and Weissbach, (1989) Methods forPlant Molecular Biology, Academic Press, and Gelvin et al. (1990) PlantMolecular Biology Manual, Kluwer Academic Publishers. Specific examplesinclude those derived from a Ti plasmid of Agrobacterium tumefaciens, aswell as those disclosed by Herrera-Estrella et al. (1983) Nature 303:209, Bevan (1984) Nucl. Acids Res. 12: 8711-8721, Klee (1985)Bio/Technology 3: 637-642. Ti-derived plasmids can be transferred intoboth monocot and dicot species using Agrobacterium-mediatedtransformation (Ishida et al (1996) Nat. Biotechnol. 14: 745-750; Bartonet al. (1983) Cell 32: 1033-1043).

[0301] Alternatively, non-Ti vectors can be used to transfer the DNAinto plant cells by using free DNA delivery techniques. Such methods mayinvolve, for example, the use of liposomes, electroporation,microprojectile bombardment, silicon carbide whiskers, and viruses. Byusing these methods transgenic plants such as wheat, rice (Christou(1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm (1990) PlantCell 2: 603-618) can be produced. An immature embryo can also be a goodtarget tissue for plants for direct DNA delivery techniques by using theparticle gun (Weeks et al. (1993) Plant Physiol. 102: 1077-1084; Vasil(1993) Bio/Technology 10: 667-674; Wan et al. (1994) Plant Physiol. 104:37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996)Nature Biotech. 14: 745-750).

[0302] Typically, plant transformation vectors include one or morecloned plant coding sequences (genomic or cDNA) under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant transformation vectors typically alsocontain a promoter (for example, a regulatory region controllinginducible or constitutive, environmentally-or developmentally-regulated,or cell- or tissue-specific expression), a transcription initiationstart site, an RNA processing signal (such as intron splice sites), atranscription termination site, and/or a polyadenylation signal.

[0303] Examples of constitutive plant promoters which may be useful forexpressing the CBF sequence include: the cauliflower mosaic virus (CaMV)35S promoter, which confers constitutive, high-level expression in mostplant tissues (see, for example, Odell et al. (1985) Nature 313:810-812); the nopaline synthase promoter (An et al. (1988) PlantPhysiol. 88: 547-552); and the octopine synthase promoter (Fromm et al.(1989) Plant Cell 1: 977-984).

[0304] A variety of plant gene promoters that regulate gene expressionin response to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner can be used for expression of the CBFs inplants, as illustrated by seed-specific promoters (such as the napin,phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697),root-specific promoters, such as those disclosed in U.S. Pat. Nos.5,618,988, 5,837,848 and 5,905,186; fruit-specific promoters that areactive during fruit ripening (such as the dru 1 promoter (U.S. Pat. No.5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and thetomato polygalacturonase promoter (Bird et al. (1988) supra),root-specific promoters, such as those disclosed in U.S. Pat. Nos.5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such asPTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active invascular tissue (Ringli et al. (1998) Plant Mol. Biol. 37: 977-988),flower-specific (Kaiser et al. (1995) Plant Mol. Biol. 28: 231-243),auxin-inducible promoters (such as that described in van der Kop et al.(1999) Plant Mol. Biol. 39: 979-990 or Baumann et al. (1999) Plant Cell11: 323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) PlantMol. Biol. 38: 743-753), promoters responsive to gibberellin (Shi et al.(1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) 38:817-825) and the like. Additional promoters are those that elicitexpression in response to light (for example, the pea rbcS-3A promoter,Kuhlemeier et al. (1989) Plant Cell 1: 471-478, and the maize rbcSpromoter, Schaffner et al. (1991) Plant Cell 3: 997-1012); wounding (forexample, wunI, Siebertz et al. (1989) Plant Cell 1: 961-968); pathogenresistance chemicals such as methyl jasmonate or salicylic acid (Gatz etal. (1997) supra). In addition, the timing of the expression can becontrolled by using promoters such as those acting at late seeddevelopment (Odell et al. (1994) Plant Physiol. 106: 447-458).

[0305] Plant expression vectors may also include RNA processing signalsthat may be positioned within, upstream or downstream of the codingsequence. In addition, the expression vectors may include additionalregulatory sequences from the 3′-untranslated region of plant genes, forexample, a 3′ terminator region to increase mRNA stability of the mRNA,such as the PI-II terminator region of potato or the octopine ornopaline synthase 3′ terminator regions.

[0306] Finally, as noted above, plant expression vectors may alsoinclude dominant selectable marker genes to allow for the readyselection of transformants. Such genes include those encoding antibioticresistance genes (for example, resistance to hygromycin, kanamycin,bleomycin, G418, streptomycin or spectinomycin) and herbicide resistancegenes (for example, phosphinothricin acetyltransferase).

[0307] The polynucleotides and polypeptides of this invention may alsobe expressed in a plant in the absence of an expression cassette bymanipulating the activity or expression level of the endogenous gene byother means. For example, by ectopically expressing a gene by T-DNAactivation tagging (Ichikawa et al. (1997) Nature 390: 698-701, Kakimotoet al. (1996) Science 274: 982-985). This method entails transforming aplant with a gene tag containing multiple transcriptional enhancers andonce the tag has inserted into the genome, expression of a flanking genecoding sequence becomes deregulated. In another example, thetranscriptional machinery in a plant may be modified so as to increasetranscription levels of a polynucleotide of the invention (See PCTPublications WO9606166 and WO 9853057 which describe the modification ofthe DNA binding specificity of zinc finger proteins by changingparticular amino acids in the DNA binding motif).

[0308] The transgenic plant may also comprise the machinery necessaryfor expressing or altering the activity of a polypeptide encoded by anendogenous gene, for example by altering the phosphorylation state ofthe polypeptide to maintain it in an activated state.

[0309] In some cases, a reduction in plant biomass or the level ofcryoprotectants may be desired. In such a case, a reduction of biomassor the cryoprotectant levels may be achieved by decreasing the levels ofCBF expression. For example, a reduction of CBF expression in atransgenic plant to modify a plant trait may be obtained by introducinginto plants antisense constructs based on the CBF cDNA. For antisensesuppression, the CBF cDNA is arranged in reverse orientation relative tothe promoter sequence in the expression vector. The introduced sequenceneed not be the full length CBF cDNA or gene, and need not be identicalto the CBF cDNA or a gene found in the plant type to be transformed.

[0310] Vectors in which RNA encoded by the CBF cDNA (or variantsthereof) is over-expressed may also be used to obtain co-suppression ofthe endogenous CBF gene in the manner described in U.S. Pat. No.5,231,020 to Jorgensen. Such co-suppression (also termed sensesuppression) does not require that the entire CBF cDNA be introducedinto the plant cells, nor does it require that the introduced sequencebe exactly identical to the endogenous CBF gene. However, as withantisense suppression, the suppressive efficiency will be enhanced as(1) the introduced sequence is lengthened and (2) the sequencesimilarity between the introduced sequence and the endogenous CBF geneis increased.

[0311] Vectors expressing an untranslatable form of the CBF mRNA mayalso be used to suppress the expression of endogenous CBF activity tomodify a trait. Methods for producing such constructs are described inU.S. Pat. No. 5,583,021 to Dougherty et al. Preferably, such constructsare made by introducing a premature stop codon into the CBF gene.Alternatively, a plant trait may be modified by gene silencing usingdouble-strand RNA (Sharp (1999) Genes Development 13: 139-141) or bysimultaneous expression of both sense and antisense RNAs (Waterhouse etal. (1998) Proc. Natl. Acad. Sci. 95: 13959-13964).

[0312] Another method for abolishing the expression of a gene is byinsertion mutagenesis using the T-DNA of Agrobacterium tumefaciens.After generating the insertion mutants, the mutants can be screened toidentify those containing the insertion in a CBF gene. Mutantscontaining a single mutation event at the desired gene may be crossed togenerate homozygous plants for the mutation (Koncz et al. (1992) Methodsin Arabidopsis Research. World Scientific).

[0313] A plant trait may also be modified by using the cre-lox system(for example, as described in U.S. Pat. No. 5,658,772). A plant genomemay be modified to include first and second lox sites that are thencontacted with a Cre recombinase. If the lox sites are in the sameorientation, the intervening DNA sequence between the two sites isexcised. If the lox sites are in the opposite orientation, theintervening sequence is inverted.

[0314] C. Transgenic Plants with Modified CBF Expression

[0315] Once an expression cassette comprising a polynucleotide encodinga CBF gene of this invention has been constructed, standard techniquesmay be used to introduce the polynucleotide into a plant in order tomodify a trait of the plant. The plant may be any higher plant,including gymnosperms, monocotyledonous and dicotyledenous plants.Suitable protocols are available for Leguminosae (alfalfa (Medicagosativa), soybean (Glycine max), clover (Trifolium), etc.), Umbelliferae(carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed(Brassica rapa L and Brassica napus L), broccoli, leaf mustard (Brassicajuncea), etc.), Curcurbitaceae (melons and cucumber), Gramineac (wheat(Triticum), corn (Zea mays), rice (Oryza sativa), barley (Hordeumvulgare), rye (Secale cereale), sorghum (Sorghum bicolor and Sorghumvulgare), millet (Panicum miliaceum, Setaria italica, and Eleusinecoracana), etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.),and various other crops. See protocols described in Ammirato et al.,editors, (1984) Handbook of Plant Cell Culture—Crop Species MacmillanPubl. Co. New York, N.Y.; Shimamoto et al. (1989) Nature 338: 274-276;Fromm et al. (1990) Bio/Technology 8: 833-839; and Vasil et al. (1990)Bio/Technology 8: 429-434.

[0316] Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is now routine, and the selection of the mostappropriate transformation technique will be determined by thepractitioner. The choice of method will vary with the type of plant tobe transformed; those skilled in the art will recognize the suitabilityof particular methods for given plant types. Suitable methods mayinclude, but are not limited to: electroporation of plant protoplasts;liposome-mediated transformation; polyethylene glycol (PEG) mediatedtransformation; transformation using viruses; micro-injection of plantcells; micro-projectile bombardment of plant cells; vacuum infiltration;and Agrobacterium tumefaciens mediated transformation. Transformationmeans introducing a nucleotide sequence in a plant in a manner to causestable or transient expression of the sequence.

[0317] Successful examples of the modification of plant characteristicsor traits by transformation with cloned sequences which serve toillustrate the current knowledge in this field of technology, and whichare herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706;5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871;5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

[0318] Following transformation, plants are preferably selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed plants, and selection of transformants can beaccomplished by exposing the plants to appropriate concentrations of theantibiotic or herbicide.

[0319] After transformed plants are selected and grown to maturity,those plants with modified plant biomass or high levels of cellprotectants are identified and used for any of the purposes describedabove. Additionally, to confirm that the modified trait is due tochanges in expression levels or activity of the polypeptide orpolynucleotide of the invention may be determined by analyzing mRNAexpression using Northern blots, RT-PCR or microarrays, or proteinexpression using immunoblots or Western blots or gel shift assays.

[0320] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLES Example 1

[0321]Arabidopsis thaliana Plant Handling Arabidopsis thaliana (L.)Heynh. ecotype Ws-2 and transgenic plants in the Ws-2 background weregrown in controlled environment chambers at 20° C. under constantillumination from cool-white fluorescent lights (100-150 μmol m⁻² s⁻¹))essentially as described (Gilmour et al. (1988) Plant Physiol.87:745-750) in Baccto planting mix (Michigan Peat, Houston, Tex.). Potswere sub-irrigated with deionized water as necessary. All seeds werecold-treated (5° C.) for 4 days immediately after planting to ensureuniform germination.

Example 2

[0322] RNA Hybridization and cDNA Probes

[0323] In the following examples, unless otherwise specified, total RNAwas extracted from Arabidopsis plants as described previously (Gilmouret al. (1988) Plant Physiol. 87: 745-750). Northern transfers wereprepared and hybridized as described (Hajela et al. (1990) PlantPhysiol. 93: 1246-1252) using high stringency wash conditions(Stockinger et al. (1997) supra). ³²P-labeled probes were prepared byrandom priming (Feinberg and Vogelstein (1983) Anal. Biochem. 132:6-13). A gene-specific probe to CBF3 was made to the 3′ end of the cDNAclone by PCR as described previously (Gilmour et al. (1998) supra).Arabidopsis cDNA clones encoding Arabidopsis sucrose synthase(182C20T7), corresponding to the SUS1 gene (Martin et al. (1993) PlantJ. 4: 367-377) and Δ′-pyrroline-5-carboxylate synthase (125M17T7),corresponding to the P5CS2 gene (Strizhov, et al. (1997) Plant J. 12:557-569), were obtained from the Arabidopsis Biological Resource Centerat Ohio State University. Probes for P5CS2 transcripts shouldcross-hybridize with the highly similar P5CS1 transcripts and thus is ameasure of total P5CS transcripts.

Example 3

[0324] Activation of Transcription in Yeast Containing C-Repeat/DREUsing CBF1, CBF2 and CBF3

[0325] This example shows that CBF1, CBF2, and CBF3 activatetranscription in yeast containing CRT/DREs upstream of a reporter gene.The CBFs were expressed in yeast under control of the ADC1 promoter on a2μ plasmid (pDB20.1; Berger et al. (1992) Cell 70: 251-265). Constructsexpressing the different CBFs were transformed into yeast reporterstrains that had the indicated CRT/DRE upstream of the lacZ reportergene. Copy number of the CRT/DREs and its orientation relative to thedirection of transcription from each promoter is indicated by thedirection of the arrow.

[0326]FIG. 15 is a graph showing transcription regulation of CRT/DREcontaining reporter genes by CBF1, CBF2, and CBF3 genes in yeast. InFIG. 15, the vertical lines across the arrows of the COR15a constructrepresent the m3cor15a mutant CRT/DRE construct. Each CRT/DRE-lacZconstruct was integrated into the URA3 locus of yeast. Error barsrepresent the standard deviation derived from three replicatetransformation events with the same CBF activator construct into therespective reporter strain. Quantitative beta-gal assays were performedas described by Rose and Botstein (Rose et al. (1983) Methods Enzymol.101: 167-180).

Example 4

[0327] Isolation and Analysis of Arabidopsis thaliana cDNA Clone (CBF1)Encoding C-Repeat/DRE Binding Factor

[0328] The following example describes the isolation of an Arabidopsisthaliana cDNA clone that encodes a C-repeat/DRE binding factor, CBF1(C-repeat/DRE Binding Factor 1). Expression of CBF1 in yeast was foundto activate transcription of reporter genes containing the C-repeat/DRE(CCGAC) as an upstream activator sequence. Meanwhile, CBF1 did notactivate transcription of mutant versions of the CCGAC binding element,indicating that CBF1 is a transcription factor that binds to theC-repeat/DRE. Binding of CBF1 to the C-repeat/DRE was also demonstratedin gel shift assays using recombinant CBF1 protein expressed inEscherichia coli. Analysis of the deduced CBF1 amino acid sequenceindicated that the protein has a potential nuclear localizationsequence, a possible acidic activation domain and an AP2 domain, aDNA-binding motif of about 60 amino acids that is similar to thosepresent in Arabidopsis proteins APETALA2, AINTEGUMENTA and TINY, thetobacco ethylene response element binding proteins, and numerous otherplant proteins of unknown function.

[0329] Cold Treatment.

[0330] Plants were treated by placing pots in a cold room at 2.5° C.under cool constant illumination with white florescent lamps (25 μmolm⁻²s⁻¹) for the indicated times.

[0331] Arabidopsis cDNA Expression Library.

[0332] The Arabidopsis pACT cDNA expression library was constructed byJohn Walker and colleagues (NSF/DOE/USDA Collaborative Research in PlantBiology Program grant USDA 92-37105-7675) and deposited in theArabidopsis Biological Resource Center (stock #CD4-10).

[0333] Yeast Reporter Strains.

[0334] Oligonucleotides (Table 4) (synthesized at the MSU MacromolecularStructure Facility) encoding either wild-type or mutant versions of theC-repeat/DRE were ligated into the BglII site of the lacZ reportervector pBgl-lacZ (Li et al. (1993) Science 262: 1870-1874); kindlyprovided by Joachim Li). The resulting reported constructs wereintegrated into the ura3 locus of Saccharomyces cerevisiae strain GGY1(MAT gal4 gal80 ura3 leu2 his3 ade2 tyr) (Li et al. (1993) supra;provided by Joachim Li) by transformation and selection for uracilprototrophy.

[0335]E. coli Strains

[0336]Escherichia coli strain GM2163 containing plasmid pEJS251 wasdeposited under the Budapest Treaty on May 17, 1996 with the AmericanType Culture Collection, Rockville, Md. as ATCC 98063. It is availableby name and number pursuant to the provisions of the Budapest Treaty.TABLE 4 Oligonucleotides encoding wild type and mutant versions of theC-repeat/DRE C-repeat/ Oligonucleotide DRE* Sequence SEQ ID NO: MT50COR15a GatcATTTCATGGCCGACCTGCTTTTT 3 MT52 M1COR15aCACAATTTCAaGaattcaCTGCTTTTTT 4 MT80 M2COR15a GatcATTTCATGGtatgtCTGCTTTTT5 MT125 M3COR15a GatcATTTCATGGaatcaCTGCTTTTT 6 MT68 COR15bGatcACTTGATGGCCGACCTCTTTTTT 7 MT66 C0R78-1 GatcAATATACTACCGACATGAGTTCT 8MT86 C0R78-2 ACTACCGACATGAGTTCCAAAAAGC 9

[0337] * The C-repeat/DRE sequences tested are either wild-type found inthe promoters of COR15a (Baker et al. (1994) Plant Mol. Biol. 24:701-713), COR15b or COR78/RD29a (Horvath et al. (1993) Plant Physiol.103: 1047-1053; Yamaguchi-Shinozaki et al. (1994) Plant Cell 6: 251-264)or are mutant versions of the COR15a C-repeat/DRE (M1COR15a, M2COR15aand M3COR15a).

[0338] # Uppercase letters designate bases in wild type C-repeat/DREsequences. The core CCGAC sequence common to the above sequences isindicated in bold type. Lowercase letters at the beginning of a sequenceindicate bases added to facilitate cloning. The lowercase letters thatare underlined indicate the mutations in the C-repeat/DRE sequence ofCOR15a.

[0339] Screen of Arabidopsis cDNA Library.

[0340] The Arabidopsis pACT cDNA expression library was screened forclones encoding C-repeat/DRE biomass and environmental stress responseregulatory elements by the following method. The cDNA library, harboredin Escherichia coli BNN132, was amplified by inoculating 0.5 ml of theprovided glycerol stock into 1 L of M9 minimal glucose medium (Sambrooket al. (1989) supra) and shaking the bacteria for 20 h at 37° C. PlasmidDNA was isolated and purified by cesium chloride density gradientcentrifugation (Sambrook et al. (1989) supra) and transformed into theyeast GGY1 reporter strains selecting for leucine prototrophy. Yeasttransformants that had been grown for 2 or 3 days at 30° C. wereoverlaid with either a nitrocellulose membrane filter (Schleicher andSchuell, Keene, N.H.) or Whatman #50 filter paper (Hillsboro, Oreg.) andincubated overnight at 30° C. The yeast impregnated filters were thenlifted from the plate and treated with X-gal(5-bromo-4-chloro-3-indolyl-D-galactosidase) to assay colonies forbeta-galactosidase activity (Li et al. (1993) supra). Plasmid DNA from“positive” transformants (those forming blue colonies on theX-gal-treated filters) was recovered (Strathern et al. (1991) MethodsEnzymol. 194: 319-329), propagated in E. coli DH5α and transformed backinto the yeast reporter strains to confirm activity.

[0341] Yeast Transformation and Quantitative Beta-galactosidase Assays.

[0342] Yeast were transformed by either electroporation (Becker et al.(1991) Methods Enzymol. 194: 182-187) or the lithium acetate/carrier DNAmethod (Schiestl et al. (1989) Current Genetics 16: 339-346).Quantitative in vitro beta-galactosidase assays were done as described(Rose et al. (1983) Methods Enzymol. 101: 167-180).

[0343] Expression of CBF1 Protein in E. coli and Yeast.

[0344] CBF1 was expressed in E. coli using the pET-28a(+) vector(Novagen, Madison, Wis.). The BglII-BclI restriction fragment of pACT-11encoding CBF1 was ligated into the BamHI site of the vector bringingCBF1 under control of the T7 phage promoter. The construct resulted in a“histidine tag,” a thrombin recognition sequence and a “T7 epitope tag”being fused to the amino terminus of CBF1. The construct was transformedinto E. coli BL21 (DE3) and the recombinant CBF1 protein was expressedas recommended by the supplier (Novagen). Expression of CBF1 in yeastwas accomplished by ligating restriction fragments encoding CBF1 (theBclI-BglII and BglII-BglII fragments from pACT-11) into the BglII siteof pDB20.1 (Berger et al. (1992) Cell 70: 251-265) bringing CBF1 undercontrol of the constitutive ADC1 (alcohol dehydrogenase constitutive 1)promoter.

[0345] Gel Shift Assays.

[0346] The presence of expressed protein that binds to a C-repeat/DREbinding domain was evaluated using the following gel shift assay. Totalsoluble E. coli protein (40 ng) was incubated at room temperature in 10μl of 1× binding buffer [15 mM HEPES (pH 7.9), 1 mM EDTA, 30 mM KCl, 5%glycerol, 5% BSA, 1 mM DTT) plus 50 ng poly(dI-dC):poly(dI-dC)(Pharmacia, Piscataway, N.J.) with or without 100 ng competitor DNA.After 10 min, probe DNA (1 ng) that was ³²P-labeled by end-filling(Sambrook et al. supra) was added and the mixture incubated for anadditional 10 min. Samples were loaded onto polyacrylamide gels (4% w/v)and fractionated by electrophoresis at 150V for 2 h (Sambrook et al.supra). Probes and competitor DNAs were prepared from oligonucleotideinserts ligated into the BamHI site of pUC118 (Vicira et al. (1987)Methods Enzymol. 153: 3-11). The orientation and concatenation number ofthe inserts were determined by dideoxy DNA sequence analysis (Sambrooket al. (1989), supra). Inserts were recovered after restrictiondigestion with EcoRI and HindIII and fractionation on polyacrylamidegels (12% w/v) (Sambrook et al. (1989) supra).

[0347] Northern and Southern Analysis.

[0348] Northern and Southern analysis was performed as follows. TotalRNA was isolated from Arabidopsis (Gilmour et al. (1988) Plant Physiol.87: 745-750) and the poly(A)⁺ fraction purified using oligo dT cellulose(Sambrook et al. (1989), supra). Northern transfers were prepared andhybridized as described (Hajela et al. (1990) Plant Physiol. 93:1246-1252) except that high stringency wash conditions were at 50 C in0.1×SSPE [1.0×SSPE is 3.6 M NaCl, 20 mM EDTA, 0.2 M Na₂—HPO₄ (pH7.7)],0.5% SDS. Membranes were stripped in 0.1×SSPE, 0.5% SDS at 95° C. for 15min prior to re-probing. Total Arabidopsis genomic DNA was isolated(Stockinger et al. (1996) J. Heredity 87: 214-218) and Southerntransfers prepared (Sambrook et al. supra) using nylon membranes (MSI,Westborough, Mass.). High stringency hybridization and wash conditionswere as described by Walling (Walling et al. (1988) Nucleic Acids Res.16: 10477-10492). Low stringency hybridization was in 6×SSPE, 0.5% SDS,0.25% low fat dried milk at 60° C. Low stringency washes were in 1×SSPE,0.5% SDS at 50° C. Probes used for the entire CBF1 coding sequence and3′ end of CBF1 were the BclI/BglII and EcoRV/BglII restriction fragmentsfrom pACT-11, respectively, that had been gel purified (Sambrook et al.(1989), supra). DNA probes were radiolabeled with ³²P-nucleotides byrandom priming (Sambrook et al. (1989), supra). Autoradiography wasperformed using hyperfilm-MP (Amersham, Arlington Heights Ill.).Radioactivity was quantified using a Betascope 603 blot analyzer(Betagen Corp., Waltham Mass.).

[0349] Screen of Arabidopsis cDNA Library for Sequence Encoding aC-Repeat/DRE Binding Domain.

[0350] The “one-hybrid” strategy (Li et al. (1993) supra) was used toscreen for Arabidopsis cDNA clones encoding a C-repeat/DRE bindingdomain. In brief, yeast strains were constructed that contained a lacZreporter gene with either wild-type or mutant C-repeat/DRE sequences inplace of the normal UAS (upstream activator sequence) of the GAL1promoter.

[0351]FIGS. 1A and 1B show how the yeast reporter strains wereconstructed. FIG. 1A is a schematic diagram showing the screeningstrategy. Yeast reporter strains were constructed that carriedC-repeat/DRE sequences as UAS elements fused upstream of a lacZ reportergene with a minimal GAL1 promoter. The strains were transformed with anArabidopsis expression library that contained random cDNA inserts fusedto the GAL4 activation domain (GAL4-ACT) and screened for blue colonyformation on X-gal-treated filters. FIG. 1B is a chart showing activityof the “positive” cDNA clones in yeast reporter strains. Theoligonucleotides (oligos) used to make the UAS elements, and theirnumber and direction of insertion, are indicated by the arrows.

[0352] Yeast strains carrying these reporter constructs produced lowlevels of beta-galactosidase and formed white colonies on filterscontaining X-gal. The reporter strains carrying the wild-typeC-repeat/DRE sequences were transformed with a DNA expression librarythat contained random Arabidopsis cDNA inserts fused to the acidicactivator domain of the yeast GAL4 transcription factor, “GAL4-ACT”(FIG. 1A). The notion was that some of the clones might contain a cDNAinsert encoding a C-repeat/DRE binding domain fused to GAL4-ACT and thatsuch a hybrid protein could potentially bind upstream of the lacZreporter genes carrying the wild type C-repeat/DRE sequence, activatetranscription of the lacZ gene and result in yeast forming blue colonieson X-gal-treated filters.

[0353] Upon screening about 2×10⁶ yeast transformants, three “positive”cDNA clones were isolated; i.e., clones that caused yeast strainscarrying lacZ reporters fused to wild-type C-repeat/DRE inserts to formblue colonies on X-gal-treated filters (FIG. 1B). The three cDNA clonesdid not cause a yeast strain carrying a mutant C-repeat/DRE fused toLacZ to turn blue (FIG. 1B). Thus, activation of the reporter genes bythe cDNA clones appeared to be dependent on the C-repeat/DRE sequence.Restriction enzyme analysis and DNA sequencing indicated that the threecDNA clones had an identical 1.8 kb insert (FIG. 2A). One of the clones,designated pACT-11, was chosen for further study.

[0354] Identification of 24 kDa Polypeptide with an AP2 Domain Encodedby pACT-11.

[0355]FIGS. 2A, 2B, 2C and 2D provide an analysis of the pACT-11 cDNAclone. FIG. 2A is a schematic drawing of the pACT-11 cDNA insertindicating the location and 5′ to 3′ orientation of the 24 kDapolypeptide and 25s rRNA sequences. The cDNA insert was cloned into theXhoI site of the pACT vector. FIG. 2B is a DNA and amino acid sequenceof the 24 kDa polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2). The AP2domain is indicated by a double underline. The basic amino acids thatpotentially act as a nuclear localization signal are indicated withasterisks. The BclI site immediately upstream of the 24 kDa polypeptideused in subcloning the 24 kDa polypeptide and the EcoRV site used insubcloning the 3′ end of CBF1 are indicated by single underlines. FIG.2C is a schematic drawing indicating the relative positions of thepotential nuclear localization signal (NLS), the AP2 domain and theacidic region of the 24 kDa polypeptide. Numbers indicate amino acidresidues. FIG. 2D is a chart showing comparison of the AP2 domain of the24 kDa polypeptide (SEQ ID NO: 10) with that of the tobacco DNA bindingprotein EREBP2 (Okme-Takagi et al. (1995) Plant Cell 7: 173-182; SEQ IDNO: 11). Identical amino acids are indicated with single lines; similaramino acids are indicated by double dots; amino acids that are invariantin AP2 domains are indicated with asterisks (Klucher et al. (1996) PlantCell 8: 137-153); and the histidine residues present in CBF1 and TINY(Wilson et al. (1996) Plant Cell 8: 659-671) that are tyrosine residuesin all other described AP2 domains are indicated with a caret. A singleamino acid gap in the CBF1 sequence is indicated by a single dot.

[0356] Our expectation was that the cDNA insert in pACT-11 would have aC-repeat/DRE binding domain fused to the yeast GAL4-ACT sequence.However, DNA sequence analysis indicated that an open reading frame ofonly nine amino acids had been added to the C-terminus of GAL4-ACT. Itseemed highly unlikely that such a short amino acid sequence couldcomprise a DNA binding domain. Also surprising was the fact that abouthalf of the cDNA insert in pACT-11 corresponded to 25s rRNA sequences(FIG. 2A). Further analysis, however, indicated that the insert had anopen reading frame, in opposite orientation to the GAL4-ACT sequence,deduced to encode a 24 kDa polypeptide (FIGS. 2A-2C). The polypeptidehas a basic region that could potentially serve as a nuclearlocalization signal (Raikhel (1992) Plant Physiol. 100: 1627-1632) andan acidic C-terminal half (pI of 3.6) that could potentially act as anacidic transcription activator domain (Hahn (1993) Cell 72: 481-483). Asearch of the nucleic acid and protein sequence databases indicated thatthere was no previously described homology of the 24 kDa polypeptide.However, the polypeptide did have an AP2 domain (Jofuku et al. (1994)Plant Cell 6: 1211-1225 (FIGS. 2B, 2D), a DNA binding motif of about 60amino acids (Ohme-Takagi et al. (1994) Plant Cell 7: 173-182) that ispresent in numerous plant proteins including the APETALA2 (Jofuku et al.(1994) Plant Cell 6: 1211-1225), AINTEGUMENTA (Klucher et al. (1996)Plant Cell 8: 137-153; Elliot et al. (1996) Plant Cell 8: 155-168) andTINY (Wilson et al. (1996) Plant Cell 8: 659-671) proteins ofArabidopsis and the EREBPs (ethylene response element binding proteins)of tobacco (Ohme-Takagi et al. (1995) Plant Cell 7: 173-182).

[0357] 24 kDa Polypeptide Binds to the C-Repeat/DRE and ActivatesTranscription in Yeast.

[0358] We hypothesized that the 24 kDa polypeptide was responsible foractivating the lacZ reporter genes in yeast. To test this, theBclI-BglII fragment of pACT-11 containing the 24 kDa polypeptide, andthe BglII-BglII fragment containing the 24 kDa polypeptide plus a smallportion of the 25s rRNA sequence, was inserted into the yeast expressionvector pDB20.1

[0359]FIG. 3 is a chart showing activation of reporter genes by the 24kDa polypeptide. Restriction fragments of pACT-11 carrying the 24 kDapolypeptide (BclI-BglII) or the 24 kDa polypeptide plus a small amountof 25s RNA sequence (BglII-BglII) were inserted in both orientationsinto the yeast expression vector pDB20.1 (see FIGS. 2A and 2B forlocation of BclI and BglII restriction sites). These “expressionconstructs” were transformed into yeast strains carrying the lacZreporter gene fused to direct repeat dimers of either the wild-typeCOR15a C-repeat/DRE (oligonucleotide MT50) or the mutant M2COR15aC-repeat/DRE (oligonucleotide MT80). The specific activity ofbeta-galactosidase (nmoles o-nitrophenol produced/min⁻¹×mg protein⁻¹)was determined from cultures grown in triplicate. Standard deviationsare indicated. Abbreviations: pADC1, ADC1 promoter; tADC 1, ADC1terminator.

[0360] Plasmids containing either insert in the same orientation as theADC1 promoter stimulated synthesis of beta-galactosidase whentransformed into yeast strains carrying the lacZ reporter gene fused toa wild-type COR15a C-repeat/DRE (FIG. 3). The plasmids did not, however,stimulate synthesis of beta-galactosidase when transformed into yeaststrains carrying lacZ fused to a mutant version of the COR15aC-repeat/DRE (FIG. 3). These data indicated that the 24 kDa polypeptidecould bind to the wild-type C-repeat/DRE and activate expression for thelacZ reporter gene in yeast. Additional experiments indicated that the24 kDa polypeptide could activate expression of the lacZ reporter genefused to either a wild-type COR78 C-repeat/DRE (dimer of MT66) or awild-type COR15b C-repeat/DRE (dimer of MT 68) (not shown). A plasmidcontaining the BclI-BglII fragment (which encodes only the 24 kDapolypeptide) cloned in opposite orientation to the ADC1 promoter did notstimulate synthesis of beta-galactosidase in reporter strains carryingthe wild-type COR15a C-repeat/DRE fused to lacZ (FIG. 3). In contrast, aplasmid carrying the BglII-BglII fragment (containing the 24 kDapolypeptide plus some 25s rRNA sequences) cloned in opposite orientationto the ADC1 promoter produced significant levels of beta-galactosidasein reporter strains carrying the wild-type COR15a C-repeat/DRE (FIG. 3).Thus, a sequence located closely upstream of the 24 kDa polypeptide wasable to serve as a cryptic promoter in yeast, a result that offered anexplanation for how the 24 kDa polypeptide was expressed in the originalpACT-11 clone.

[0361] Gel Shift Analysis Indicates that the 24 kDa Polypeptide Binds tothe C-Repeat/DRE.

[0362] Gel shift experiments were conducted to demonstrate further thatthe 24 kDa polypeptide bound to the C-repeat/DRE. Specifically, the openreading frame for the 24 kDa polypeptide was inserted into thepET-28a(+) bacterial expression vector and the resulting 28 kDa fusionprotein was expressed at high levels in E. coli. (FIG. 4).

[0363]FIG. 4 is a photograph of an electrophoresis gel showingexpression of the recombinant 24 kDa polypeptide in E. coli. Shown arethe results of SDS-PAGE analysis of protein extracts prepared from E.coli harboring either the expression vector alone (vector) or the vectorplus an insert encoding the 24 kDa polypeptide in sense (sense insert)or antisense (antisense insert) orientation. The 28 kDa fusion protein(see Materials and Methods) is indicated by an arrow.

[0364]FIG. 5 is a photograph of a gel for shift assays indicating thatCBF1 binds to the C-repeat/DRE. The C-repeat/DRE probe (1 ng) used inall reactions was a ³²P-labeled dimer of the oligonucleotide MT50 (wildtype C-repeat/DRE from COR15a). The protein extracts used in the firstfour lanes were either bovine serum albumin (BSA) or the indicated CBF1sense, antisense and vector extracts described in FIG. 4. The eightlanes on the right side of the figure used the CBF1 sense proteinextract plus the indicated competitor C-repeat/DRE sequences (100 ng).The numbers 1×, 2× and 3× indicate whether the oligonucleotides weremonomers, dimers or trimers, respectively, of the indicated C-repeat/DREsequences.

[0365] Protein extracts prepared from E. coli expressing the recombinantprotein produced a gel shift when a wild-type COR15a C-repeat/DRE wasused as probe (FIG. 5). No shift was detected with BSA or E. coliextracts prepared from strains harboring the vector alone, or the vectorwith an antisense insert for the 24 kDa polypeptide. Oligonucleotidesencoding wild-type C-repeat/DRE sequences from COR15a or COR78 competedeffectively for binding to the COR15a C-repeat/DRE probe, but mutantversion of the COR15a C-repeat/DRE did not (FIG. 5). These in vitroresults corroborated the in vivo yeast expression studies indicatingthat the 24 kDa polypeptide binds to the C-repeat/DRE sequence. The 24kDa polypeptide was thus designated CBF1 (C-repeat/DRE binding factor 1)and the gene encoding it named CBF1.

[0366] CBF1 is a Unique or Low Copy Number Gene.

[0367]FIG. 6 is a photograph of a southern blot analysis indicating CBF1is a unique or low copy number gene. Arabidopsis DNA (1 μg) was digestedwith the indicated restriction endonucleases and southern transfers wereprepared and hybridized with a ³²P-labeled probe encoding the entireCBF1 polypeptide.

[0368] The hybridization patterns observed in southern analysis ofArabidopsis DNA using the entire CBF1 gene as probe were relativelysimple indicating that CBF1 is either a unique or low copy number gene(FIG. 6). The hybridization patterns obtained were not altered if onlythe 3′ end of the gene was used as the probe (the EcoRV/BglIIrestriction fragment from pACT-11 encoding the acidic region of CBF1,but not the AP2 domain) or if hybridization was carried out at lowstringency (not shown).

[0369] CBF1 Transcript Level Response to Low Temperature.

[0370]FIGS. 7A, 7B and 7C relate to CBF1 transcripts in control andcold-treated Arabidopsis. FIG. 7A is a photograph of a membrane RNAisolated from Arabidopsis plants that were grown at 22° C. or grown at22° C. and transferred to 2.5° C. for the indicated times. FIGS. 7B and7C are graphs showing relative transcript levels of CBF1 and COR15a incontrol and cold-treated plants. The radioactivity present in thesamples described in FIG. 7A were quantified using a Betascope 603 blotanalyzer and plotted as relative transcript levels (the values for the22° C. grown plants being arbitrarily set as 1) after adjusting fordifferences in loading using the values obtained with the pHH25 probe.

[0371] Based on the original data used to prepare FIGS. 7A-7C, northernanalysis indicated that the level of CBF1 transcripts increased about 2to 3 fold in response to low temperature (FIG. 7B). Following thisanalysis, it was determined that CBF genes are induced rapidly (within15 min of treatment) by mechanical agitation as well as coldtemperatures (Gilmour et al. (1998) Plant J. 16: 433-443), and that CBFgenes are not expressed at significant levels in non-stressed plants.The detectable level of CBF1 transcripts at the “0” time point in FIGS.7A and 7B was subsequently shown to be due to agitation, and that CBF1transcript levels actually increased significantly more in response tolow temperature than 2 to 3 fold over control plants, when agitation waseliminated as a inducer of CBF genes in all of the plants.

[0372] The transcript levels for COR15a increased approximately 35 foldin cold-treated plants (FIG. 7C). Only a singly hybridizing band wasobserved for CBF1 at either high or low stringency with probes foreither the entire CBF1 coding sequence or the 3′ end of the gene (theEcoRV/BglII fragment of pACT-11) (not shown). The size of the CBF1transcripts was about 1.0 kb.

[0373] The above example regarding CBF1 represents the firstidentification of a gene sequence that encodes a protein capable ofbinding to the C-repeat/DRE sequence CCGAC. The experimental resultspresented evidence that CBF1 binds to the C-repeat/DRE both in vitro viagel shift assays and in vivo via yeast expression assays. Further, theresults demonstrate that CBF1 can activate transcription of reportergenes in yeast that contain the C-repeat/DRE.

[0374] The results of the southern analysis indicate that CBF1 is aunique or low copy number gene in Arabidopsis. However, the CBF1 proteincontains a 60 amino acid motif, the AP2 domain that is evolutionaryconserved in plants (Weigel (1995) Plant Cell 7: 388-389). It is presentin the APETALA2 (Jofuku et al. (1994) Plant Cell 6: 1211-1225),AINTEGUMENTA (Klucher et al. (1996) Plant Cell 8: 137-153; and Elliot etal. (1996) Plant Cell 8: 155-168), TINY (Wilson et al. Plant Cell 8:659-671 (1996)) and cadmium-induced (Choi et al. (1995) Plant Physiol.108: 849) proteins of Arabidopsis and the EREBPs of tobacco (Ohme-Takagiet al. (1995) Plant Cell 7: 173-182). In addition, a search of theGenBank expressed sequence tagged cDNA database indicates that there isone cDNA from B. napus, two from Ricinus communis, and more than 25 fromArabidopsis and 15 from rice, that are deduced to encode proteins withAP2 domains. The results of Ohme-Takagi and Shinshi (Ohme-Takagi et al.supra) indicate that the function of the AP2 domain is DNA-binding; thisregion of the putative tobacco transcription factor EREBP2 isresponsible for its binding to the cis-acting ethylene response elementreferred to as the GCC-repeat. As discussed by Ohme-Takagi and Shinshi(Ohme-Takagi et al. supra), the DNA-binding domain of EREBP2 (the AP2domain) contains no significant amino acid sequence similarities orobvious structural similarities with other known transcription factorsor DNA binding motifs. Thus, the domain appears to be a novelDNA-binding motif that to date, has only been found in plant proteins.

[0375] It is generally believed that that the CCGAC core sequence is amember of family of core sequences having the common subsequence CCG,and that the binding of CBF1 to the C-repeat/DRE involves the AP2domain. In this regard, it is germane to note that the tobacco ethyleneresponse element, AGCCGCC, closely resembles the C-repeat/DRE sequencespresent in the promoters of the Arabidopsis genes COR15a, GGCCGAC, andCOR78/RD29A, TACCGAC. Applicants believe that CBF1, the EREBPs and otherAP2 domain proteins are members of a superfamily of DNA binding proteinsthat recognize a family of cis-acting regulatory elements having CCG asa common core sequence. Differences in the sequence surrounding the CCGcore element could result in recruitment of different AP2 domainproteins which, in turn, could be integrated into signal transductionpathways activated by different environmental, hormonal anddevelopmental cues. Such a scenario is akin to the situation that existsfor the ACGT-family of cis-acting elements (Foster et al. (1994) FASEBJ. 8: 192-200). In this case, differences in the sequence surroundingthe ACGT core element result in the recruitment of different bZIPtranscription factors involved in activating transcription in responseto a variety of environmental and developmental signals. Applicantsbelieve that other C-repeat/DRE regulatory sequences exist which belongto a broader CCG family of regulatory sequences. By screening plantgenomes according to the methodology taught herein using other membersof the CCG family, additional regulatory sequences as well as thebinding proteins which bind to these regulatory sequences can beidentified. For example, plants which are known to have modified biomassor exhibit a form of environmental stress tolerance can be screenedaccording to the blue colony assay and other screening methodologiesused in the present invention with other members of the CCG family inorder to identify other binding proteins and their gene sequences.Examples of other members of the CCG family include, but are not limitedto, biomass or environmental stress response regulatory elements whichinclude one of the following sequences: CCGAA, CCGAT, CCGAC, CCGAG,CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT,CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG,CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT,ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG,GCCGA, GCCGT, GCCGC, and GCCGG (see U.S. Pat. No. 6,417,428).

[0376] The results of the yeast transformation experiments indicate thatCBF1 has a domain that can serve as a transcriptional activator. Themost likely candidate for this domain is the acidic C-terminal half ofthe polypeptide. Indeed, random acidic amino acid peptides from E. colihave been shown to substitute for the GAL4 acidic activator domain ofGAL4 in yeast (Ma et al. (1987) Cell 51: 113-199). Moreover, acidicactivator domains have been found to function across kingdoms (Hahn(1993) Cell 72: 481-483); the yeast GAL4 acidic activator, for instance,can activate transcription in tobacco (Ma et al. (1988) Nature 334:631-633). It has also been shown that certain plant transcriptionfactors, such as Vp1 (McCarty et al. (1991) Cell 66: 895-905), haveacidic domains that function as transcriptional activators in plants.Significantly, the acidic activation domains of the yeast transcriptionfactors VP16 and GCN4 require the “adaptor” proteins ADA2, ADA3, andGCN5 for full activity (see Guarente (1995) Trends Biochem. Sci. 20:517-521). These proteins form a heteromeric complex (Horiuchi et al.(1995) Mol. Cell Biol. 15: 1203-1209) that bind to the relevantactivation domains. The precise mechanism of transcriptional activationis not known, but appears to involve histone acetylation: there is awealth of evidence showing a positive correlation between histoneacetylation and the transcriptional activity of chromatin (Wolffe (1994)Trends Biochem. Sci. 19: 240-244) and recently, the GCN5 protein hasbeen shown to have histone acetyltransferase activity (Brownell et al.(1996) Cell 84: 843-851). Genetic studies indicate that CBF1, like VP16and GCN4, requires ADA2, ADA3 and GCN5 to function optimally in yeast.The fundamental question thus raised is whether plants have homologs ofADA2, ADA3 and GCN5 and whether these adaptors are required for CBF1function (and function of other transcription factors with acidicactivator regions) in Arabidopsis.

Example 5

[0377] Identification of Modified Phenotypes in Overexpression or GeneKnockout Plants

[0378] Experiments were performed to identify those transformants orknockouts that exhibited modified cell protectant levels. Among thebiochemicals that were assayed were sugars, proline and fatty acids.

[0379] Proline levels in leaves were measured by preparing lyophilizedleaf material; 30 mg samples of the lyophilized material were thenextracted with 3 ml deionized water at 80° C. for 15 min. The sampleswere shaken for approximately 1 hour at room temperature and thenallowed to stand overnight at 4° C. The extracts were filtered throughglass wool and analyzed for proline content using the acid ninhydrinreaction (Troll and Lindsley (1955) J. Biol. Chem. 215:655-660). Prolinelevels in certain samples were confirmed by amino acid analysis using anamino acid analyzer at the Macromolecular Structure Facility in theBiochemistry Department at Michigan State University.

[0380] Total soluble sugars (for example, sucrose, glucose, and fructoseamong others) were extracted from lyophilized leaf material (20 mg) in80% ethanol (2 ml) at 80° C. for 15 min. The samples were shaken forapproximately 1 hr at room temperature and allowed to stand overnight at4° C. Extracts were filtered through glass wool and chlorophyll removedby shaking samples (0.4 ml) with water (0.4 ml) and chloroform (0.4 ml).The aqueous extract was tested for sugar content using thephenol-sulfuric acid assay (Dubois et al. (1956) Anal. Chem.28:350-356). Certain samples were dried down, suspended in water and thesugars analyzed by HPLC using a sugar column (Shodex, Shoko Co. Ltd.,Japan) with a refractive index detector as previously described (Gao etal. (1999) Physiol. Plant 106:1-8). Retention times were compared tothose of standard glucose, fructose and sucrose, and the peaksintegrated using Millennium-32 software (Waters Corp.).

[0381] The fatty acid composition of plant cells and tissues may bealtered by transcriptional control of fatty acid biosynthesis. Thepresently disclosed transcription factors and variants thereof may beable to modify the expression of fatty acid biosynthetic pathways, whichmay, in turn, alter cell protectant levels within a plant. A number ofindividual fatty acids in the leaves of transgenic plants are presentlyof interest as cell protectants. These fatty acids of interest representeither end products or intermediates within one or more biosyntheticpathways. Modifications of the levels of intermediates generally affectthe throughput and yield of an entire pathway, and thus measurements ofindividual fatty acid metabolites are often representative of changes toan entire biosynthetic pathway. For example, malonyl-CoA is a commonintermediate in fatty acid biosynthesis of anthroquinones, cuticularwaxes, flavonoids, and fatty acids. The formation of malonyl-CoA may beregulated by the action of acetyl-CoA carboxylase, which catalyzesATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. Control ofmalonyl-CoA synthesis at the enzymatic or transcriptional level has aprofound impact on various fatty acid levels via a number of pathways(Higuchi, T. (1997) Biochemistry and Molecular Biology of Wood,Springer-Verlag, Berlin, pages 238-242, and Buchanan et al (2000)Biochemistry and Molecular Biology of Plants, American Society of PlantPhysiology, Rockville, Md., pages 465-471) for example, saturated fattyacids such as 18:1, 18:2 and/or 18:3 fatty acids that may protect plantsfreezing stress). Thus, analysis of both total lipids and of individualfatty acids that affect entire pathways is central to understanding howfatty acid biosynthesis and composition can act to modulate a plant'scell protectant levels.

[0382] Total lipids from Arabidopsis leaves were measured by extraction,hydrolysis, and methylation essentially as described by Benning andSomerville ((1992) J. Bacteriol. 174: 2352-2360). Triplicate samples ofleaf material (approximately 20 mg fresh weight) were placed inTEFLON-lined glass screw cap tubes with 1 ml 1N HCl in methanol andheated at 80° C. for 40 min. Myristic acid (14:0) (5 μg) was added as aninternal standard to each sample. The resulting fatty acid methyl esterswere partitioned into 0.9% NaCl in hexane (1 ml), the hexane phaseconcentrated to a small volume and the entire sample separated by gaschromatography as detailed (Rossak et al. 1997 Arch. Biochem. Biophys.340:219-230). The individual fatty acids were quantified using AGP_TOPsoftware (Hewlett Packard). Experiments were performed to identify thosetransformants or knockouts that exhibited an improved environmentalstress tolerance, including cold or freezing stress, drought stress orsalt stress, as described in the following examples. For such studies,the transformants were exposed to a variety of environmental stresses.Plants were exposed to chilling stress (6 hour exposure to 4-8° C.),heat stress (6 hour exposure to 32-37° C.), high salt stress (6 hourexposure to 200 mM NaCl), drought stress (168 hours after removing waterfrom trays), or osmotic stress (6 hour exposure to 3 M mannitol).

Example 6

[0383] Use of CBF1 to Induce Cold Regulated Gene Expression in Non-ColdAcclimated Arabidopsis Plants.

[0384] The following example demonstrates that increased expression ofCBF1 induces COR gene expression in non-cold acclimated Arabidopsisplants. Transgenic Arabidopsis plants that overexpress CBF1 were createdby placing a cDNA encoding CBF1 under the control of the strongcauliflower mosaic virus (CaMV) 35S promoter and transforming thechimeric gene into Arabidopsis ecotype RLD plants (Standard procedureswere used for plasmid manipulations (Sambrook et al. (1989), supra). TheCBF1-containing AseI-BglII fragment from pACT-Bgl+ (Stockinger et al.(1997) Proc. Natl. Acad. Sci. 94: 1035) was gel-purified, BamHI linkerswere ligated to both ends and the fragment was inserted into the BamHIsite in pCIB710 (Rothstein et al. (1987) Gene 53: 153-161) whichcontains the CaMV 35S promoter and terminator. The chimeric plasmid waslinearized at the KpnI site and inserted into the KpnI site of thebinary vector pCIB10g (Ciba-Geigy, Research Triangle Park, N.C.). Theplasmid was transformed into Agrobacterium tumefaciens strain C58C1(pMP90) by electroporation. Arabidopsis plants were transformed by thevacuum infiltration procedure (Bechtold et al. (1993) Acad. Sci. Paris,Life Sci. 316: 1194-1199) as modified (van Hoof et al. (1996) PlantJournal 10: 415-424). Initial screening gave rise to two transgeniclines, A6 and B16, that accumulated CBF1 transcripts at elevated levels.

[0385]FIG. 8 is a Northern blot showing CBF1 and COR transcript levelsin RLD and transgenic Arabidopsis plants. Leaves from non-coldacclimated and three-day cold-acclimated plants (Arabidopsis thalianaecotype RLD plants were grown in pots under continuous light (100μE/m²/sec) at 22 C for 18-25 days as described (Gilmour et al. PlantPhysiol. 87: 735 (1988)). In some cases, plants were thencold-acclimated by placing them at 2.5° C. under continuous light (50μE/m²/sec) for varying amounts of time. Leaves were harvested and totalRNA prepared and analyzed for CBF1 and COR transcripts by RNA blotanalysis using ³²P-radiolabeled probes (Total RNA was isolated fromplant leaves and subjected to RNA blot analysis using high stringencyhybridization and wash conditions as described (Stockinger et al. (1997)Proc. Natl. Acad. Sci. 94: 1035-1040; and Gilmour et al. (1988) PlantPhysiol. 87: 745-750).

[0386]FIG. 9 is an immunoblot showing COR15am protein levels in RLD andtransgenic Arabidopsis plants. Total soluble protein (100 μg) wasprepared from leaves of the non-cold acclimated RLD (RLDw), 4-daycold-acclimated RLD (RLDc4), 7-day cold-acclimated RLD (RLDc7) andnon-cold acclimated A6 and B16 plants and the levels of COR15amdetermined by immunoblot analysis using antiserum raised against theCOR15am polypeptide (Total soluble protein was isolated from plantleaves, fractionated by tricine SDS-PAGE and transferred to 0.2 micronnitrocellulose as previously described (Artus et al. (1996) Proc. Natl.Acad. Sci. 93: 13404). COR15am protein was detected using antiserumraised to purified COR15am and protein A conjugated alkaline phosphatase(Sigma-Aldrich, St. Louis, Mo.) (Artus et al. (1996) supra). No reactingbands were observed with pre-immune serum (not shown).

[0387] Southern analysis indicated that the A6 line had a single DNAinsert while the B16 line had multiple inserts (not shown). Examinationof fourth generation homozygous A6 and B16 plants indicated that CBF1transcript levels were higher in non-cold acclimated A6 and B16 plantsthan they were in non-cold acclimated RLD plants, the levels in A6 beingabout three fold higher than in B16 (FIG. 8).

[0388] CBF1 overexpression resulted in strong induction of COR geneexpression (FIG. 8). Specifically, the transcript levels of COR6.6,COR15a, COR47 and COR78 were dramatically elevated in non-coldacclimated A6 and B16 plants as compared to non-cold acclimated RLDplants. The effect was greater in the A6 line, where COR transcriptlevels in non-cold acclimated plants approximated those found incold-acclimated RLD plants. The finding that COR gene expression wasgreater in A6 plants than in B16 plants was consistent with CBF1transcript levels being higher in the A6 plants (FIG. 7A). Immunoblotanalysis indicated that the levels of the COR15am (FIG. 9) and COR6.6(not shown) polypeptides were also elevated in the A6 and B16 lines, thelevel of expression again being higher in the A6 line. Attempts toidentify the CBF1 protein in either RLD or transgenic plants wereunsuccessful. Overexpression of CBF1 had no effect on the transcriptlevels for eIF4A (eukaryotic initiation factor 4A) (Metz et al. (1992)Gene 120: 313-314), a constitutively expressed gene that is notresponsive to low temperature (FIG. 8) and had no obvious effects onplant growth and development.

[0389] The results from this example demonstrate that overexpression ofthe Arabidopsis transcriptional activator CBF1 induces expression of anArabidopsis COR “regulon” composed of genes carrying the CRT/DRE DNAregulatory element. It appears that CBF1 binds to the CRT/DRE DNAregulatory elements present in the promoters of these genes andactivates transcription that is consistent with the notion of CBF1having a role in COR gene regulation. Significantly, there was a strongcorrelation between CBF1 transcript levels and the magnitude of COR geneinduction in non-cold acclimated A6, B16, and RLD plants (FIG. 8).However, upon low temperature treatment the level of CBF1 transcriptsremained relatively low in RLD plants, while COR gene expression wasinduced to about the same level as that in non-cold acclimated A6 plants(FIG. 8). Thus, it appears that CBF1 or an associated protein becomes“activated” in response to low temperature.

Example 7

[0390] CBF Overexpression Resulted in a Marked Increase in PlantFreezing Tolerance

[0391] A. Experiments with Aribidopsis thaliana

[0392] The following example describes a comparison of the freezingtolerance of non-cold acclimated Arabidopsis plants that overexpressCBF1 to that of cold-acclimated wild-type plants. As described below,the freezing tolerance of non-cold acclimated Arabidopsis plantsoverexpressing CBF1 significantly exceeded that of non-cold acclimatedwild-type Arabidopsis plants and approached that of cold-acclimatedwild-type plants.

[0393] Freezing tolerance was determined using the electrolyte leakagetest (Sukumaran (1972) et al. Hort. Science 7: 467). Detached leaveswere frozen to various subzero temperatures and, after thawing, cellulardamage (due to freeze-induced membrane lesions) was estimated bymeasuring ion leakage from the tissues.

[0394]FIGS. 10A and 10B are graphs showing freezing tolerance of leavesfrom RLD and transgenic Arabidopsis plants. Leaves from non-coldacclimated RLD (RLDw) plants, cold-acclimated RLD (RLDc) plants andnon-cold acclimated A6, B16 and T8 plants were frozen at the indicatedtemperatures and the extent of cellular damage was estimated bymeasuring electrolyte leakage (Electrolyte leakage tests were conductedas described (Sukumaran et al. (1972) supra; and Gilmour et al. (1988)Plant Physiol. 87: 735) with the following modifications. Detachedleaves (2-4) from non-cold acclimated or cold-acclimated plants wereplaced in a test tube and submerged for 1 hour in a −2° C.water-ethylene glycol bath in a completely randomized design, afterwhich ice crystals were added to nucleate freezing. After an additionalhour of incubation at −2° C., the samples were cooled in decrements of1° C. each hour until −8° C. was reached. Samples (five replicates foreach data point) were thawed overnight on ice and incubated in 3 mldistilled water with shaking at room temperature for 3 hours.Electrolyte leakage from leaves was measured with a conductivity meter.The solution was then removed, the leaves frozen at −80° C. (for atleast one hour), and the solution returned to each tube and incubatedfor 3 hours to obtain a value for 100% electrolyte leakage. In FIG. 10Aand 10B, the RLDc plants were cold-acclimated for 10 and 11 days,respectively. Error bars indicate standard deviations.

[0395] As can be seen from FIG. 10A and 10B, CBF1 overexpressionresulted in a marked increase in plant freezing tolerance. Theexperiment presented in FIG. 10A indicates that the leaves from bothnon-cold acclimated A6 and B16 plants were more freezing tolerant thanthose from non-cold acclimated RLD plants. Indeed, the freezingtolerance of leaves from non-cold acclimated A6 plants approached thatof leaves from cold-acclimated RLD plants. The results also indicatethat the leaves from non-cold acclimated A6 plants were more freezingtolerant than those from non-cold acclimated B16 plants, a result thatis consistent with the greater level of CBF1 and COR gene expression inthe A6 line.

[0396] The results presented in FIG. 10B further demonstrate that thefreezing tolerance of leaves from non-cold acclimated A6 plants wasgreater than that of leaves from non-cold acclimated RLD plants and thatit approached the freezing tolerance of leaves from cold-acclimated RLDplants. In addition, the results indicate that overexpression of CBF1increases freezing tolerance to a much greater extent thanoverexpressing COR15a alone. This conclusion comes from comparing thefreezing tolerance of leaves from non-cold acclimated A6 and T8 plants(FIG. 10B). T8 plants (Artus (1996) supra) are from a transgenic linethat constitutively expresses COR15a (under control of the CaMV 35Spromoter) at about the same level as in A6 plants (FIG. 1). However,unlike in A6 plants, other CRT/DRE-regulated COR genes are notconstitutively expressed in T8 plants (FIG. 8).

[0397] A comparison of EL₅₀ values (the freezing temperature thatresults in release of 50% of tissue electrolytes) of leaves from RLD,A6, B16 and T8 plants is presented in Table 5.

[0398] EL₅₀ values were calculated and compared by analysis of variancecurves fitting up to third order linear polynomial trends weredetermined for each electrolyte leakage experiment. To insure unbiasedpredictions of electrolyte leakage, trends significantly improving themodel fit at the 0.2 probability level were retained. EL₅₀ values werecalculated from the fitted models. In Table 2, an unbalanced one-wayanalysis of variance, adjusted for the different numbers of EL₅₀ valuesfor each plant type, was determined using SAS PROC GLM [SAS Institute,Inc. (1989), SAS/STAT User's Guide, Version 6, Cory, N.C.)]. EL₅₀values±SE (n) are presented on the diagonal line for leaves fromnon-cold acclimated RLD (RLDw), cold-acclimated (7 to 10 days) RLD(RLDc) and non-cold acclimated A6, B16 and T8 plants. P values forcomparisons of EL₅₀ values are indicated in the intersecting cells.TABLE 5 EL₅₀ values RLDw RLDc A6 B16 T8 RLDw −3.9 ± 0.21 (8) P < 0.0001P < 0.0001 P = 0.0014 P = 0.7406 RLDc −7.6 ± 0.30 (4) P = 0.3261 P <0.0001 P < 0.0001 A6 −7.2 ± 0.25 (6) P < 0.0001 P < 0.0001 B16 −5.2 ±0.27 (5) P = 0.0044 T8 −3.8 ± 0.35 (3)

[0399] The data confirm that: 1) the freezing tolerance of leaves fromboth non-cold acclimated A6 and B16 plants is greater than that ofleaves from both non-cold acclimated RLD and T8 plants; and 2) thatleaves from non-cold acclimated A6 plants are more freezing tolerantthan leaves from non-cold acclimated B16 plants. No significantdifference was detected in EL₅₀ values for leaves from non-coldacclimated A6 and cold-acclimated RLD plants or from non-cold acclimatedRLD and T8 plants.

[0400] The enhancement of freezing tolerance in the A6 line was alsoapparent at the whole plant level. FIG. 11 is a photograph showingfreezing survival of RLD and A6 Arabidopsis plants. Non-cold acclimated(WARM) RLD and A6 plants and 5-day cold-acclimated (COLD) RLD plantswere frozen at −5° C. for 2 days and then returned to a growth chamberat 22° C. (Pots (3.5 inch) containing about 40 non-cold acclimatedArabidopsis plants (20 day old) and 4 day cold-acclimated plants (25days old) (Arabidopsis thaliana ecotype RLD plants were grown in potsunder continuous light (100 μE/m²/sec) at 22° C. for 18-25 days asdescribed (Gilmour et al. (1988) Plant Physiol. 87: 735). In some cases,plants were then cold-acclimated by placing them at 2.5° C. undercontinuous light (50 μE/m²/sec) for varying amounts of time) were placedin a completely randomized design in a −5° C. cold chamber in the dark.After 1 hour, ice chips were added to each pot to nucleate freezing.Plants were removed after 2 days and returned to a growth chamber at 22°C.). A photograph of the plants after 7 days of regrowth is shown.

[0401] Although the magnitude of the difference varied from experimentto experiment, non-cold acclimated A6 plants consistently displayedgreater freezing tolerance in whole plant freeze tests than did non-coldacclimated RLD plants (FIG. 11). No difference in whole plant freezesurvival was detected between non-cold acclimated B16 and RLD plants ornon-cold acclimated T8 and RLD plants (not shown).

[0402] The results of this experiment show that CBF1-induced expressionof CRT/DRE-regulated COR genes result in a dramatic increase in freezingtolerance and confirms the belief that COR genes play a major role inplant cold acclimation. The increase in freezing tolerance brought aboutby expressing the battery of CRT/DRE-regulated COR genes was muchgreater than that brought about by overexpressing COR15a aloneindicating that COR genes in addition to COR15a have roles in freezingtolerance.

[0403] Traditional plant breeding approaches have met with limitedsuccess in improving the freezing tolerance of agronomic plants(Thomashow (1990) Adv. Genet 28: 99-131). For instance, the freezingtolerance of the best wheat varieties today is essentially the same asthe most freezing-tolerance varieties developed in the early part of the20^(th) Century. Thus, in recent years there has been considerableinterest that biotechnology might offer new strategies to improve thefreezing tolerance of agronomic plants. By the results of the presentinvention, Applicants demonstrate the ability to enhance the freezingtolerance of non-cold acclimated Arabidopsis plants by increasing theexpressing of the Arabidopsis regulatory gene CBF1. As describedthroughout this application, the ability of the present invention tomodify the expression of environmental stress tolerance genes such asCOR genes has wide ranging implications since the CRT/DRE DNA regulatoryelement is not limited to Arabidopsis (Jiang et al. (1996) Plant Mol.Biol. 30: 679-684). Rather, CBF1 and homologous genes can be used tomanipulate expression of CRT/DRE-regulated COR genes in important cropspecies and thereby improve their freezing tolerance. By transformingmodified versions of CBF1 (or homologs) into such plants, it will extendtheir safe growing season, increase yield and expand areas ofproduction.

[0404] B. Experiments with Lycopersicon esculentum

[0405] Two CBF sequences were isolated from tomato and were transformedinto Arabidopsis plants using the methods described herein (Examples 4and 6). These tomato CBF-related polypeptides, LeCBF1 (SEQ ID NO: 331)and LeCBF2 (SEQ ID NO: 332), share amino acid sequences with SEQ ID NO:6(SEQ ID NO: 2; see FIG. 35). Similar freezing tolerance leakageexperiments to those conducted with Arabidopsis plants, as describedabove in Example 7a, demonstrated that CBF-induced expression ofCRT/DRE-regulated COR genes from tomato are endogenously induced whenwild-type tomato plants are exposed to cold-stress (FIG. 36). Whentomato CBF1 was expressed as a transgene in Arabidopsis plants,expression of COR genes, including both mRNA expression and proteinexpression, was activated (FIG. 37). This overexpression resulted in adramatic increase in freezing tolerance, as measured by percentageelectrolyte leakage (FIG. 38).

[0406] The results of this experiment show that CBF1-induced expressionof CRT/DRE-regulated COR genes result in a dramatic increase in freezingtolerance and confirms the belief that COR genes play a major role inplant cold acclimation. The increase in freezing tolerance brought aboutby expressing the battery of CRT/DRE-regulated COR genes was muchgreater than that brought about by overexpressing COR15a aloneindicating that COR genes in addition to COR15a have roles in freezingtolerance.

[0407] Traditional plant breeding approaches have met with limitedsuccess in improving the freezing tolerance of agronomic plants(Thomashow (1990) Adv. Genet 28: 99-131). For instance, the freezingtolerance of the best wheat varieties today is essentially the same asthe most freezing-tolerance varieties developed in the early part of the20^(th) Century. Thus, in recent years there has been considerableinterest that biotechnology might offer new strategies to improve thefreezing tolerance of agronomic plants. By the results of the presentinvention, Applicants demonstrate the ability to enhance the freezingtolerance of non-cold acclimated Arabidopsis plants by increasing theexpressing of the Arabidopsis regulatory gene CBF1. As describedthroughout this application, the ability of the present invention tomodify the expression of environmental stress tolerance genes such asCOR genes has wide ranging implications since the CRT/DRE DNA regulatoryelement is not limited to Arabidopsis (Jiang et al. (1996) Plant Mol.Biol. 30: 679-684). Rather, CBF1 and homologous genes can be used tomanipulate expression of CRT/DRE-regulated COR genes in important cropspecies and thereby improve their freezing tolerance. By transformingmodified versions of CBF1 (or homologs) into such plants, it will extendtheir safe growing season, increase yield and expand areas ofproduction.

[0408] B. Experiments with Lycopersicon esculentum

[0409] CBF sequences were isolated from tomato and were transformed intoArabidopsis plants using the methods described herein (Examples 4 and6). One tomato CBF (CBF1; SEQ ID NO: 331) shares 56 % identity (121/215aminom acid residues) with SEQ ID NO:2 (G40). Similar freezing toleranceleakage experiments to those conducted with Arabidopsis plants, asdescribed above in Example 7a, demonstrate that CBF-induced expressionof CRT/DRE-regulated COR genes from tomato are endogenously induced whenwild-type tomato plants are exposed to cold-stress. When tomato CBF1 isexpressed as a transgene in Arabidopsis plants, expression of COR genes,including both mRNA expression and protein expression, is activated.This overexpression results in a dramatic increase in freezingtolerance, as measured by percentage electrolyte leakage.

[0410] These experiments demonstrate that CBF genes in diverse speciesplay a major role in plant cold acclimation.

Example 8

[0411] Selection of Promoters to Control Expression of CBF1 in Plants.

[0412] The following examples describe the isolation of differentpromoters from plant genomic DNA, construction of the plasmid vectorscarrying the CBF1 gene and the inducible promoters, transformation ofArabidopsis cells/plants with these constructs, and regeneration oftransgenic plants that have altered biomass or increased tolerance toenvironmental stresses compared to non-transformed plants

[0413] Isolation of inducible promoters from plant genomic DNAs.Inducible promoters from different plant genomic DNAs were identifiedand isolated by PCR amplification using primers designed to flank thepromoter region and contain suitable restriction sites for cloning intothe expression vector. The following genes were used to BLAST searchGenBank to find the inducible promoters: Dreb2a; P5CS; Rd22; Rd29a;Rd29b; Rab18; Cor47. Table 6 lists the accession numbers and positionsof these promoters. Table 7 lists the forward and reverse primers thatwere used to isolate the promoters. TABLE 6 Gene Name Accession No.Position Length (bps) Dreb2a AB010692 51901-53955 2054 P5CS AC00300045472-47460 1988 Rd22 D10703  17-1046 1029 Rd29a D13044 3870-5511 1641Rd29b D13044  90-1785 1695 Rab18 AB013389 8070-9757 1687 Cor47 AB004872  1-1370 1370

[0414] TABLE 7 SEQ Promoter name Primer name Cloning sites ID NO: Dreb2aDreb2a-reverse HindIII (AAGCTT) 19 Dreb2a-forward BglII (AGATCT) 20 P5CSP5CS-reverse HindIII (AAGCTT) 21 P5CS-forward BglII (AGATCT) 22 Rd22Rd22-reverse HindIII (AAGCTT) 23 Rd22-forward KpnI (GGTACC) 24 Rd29aRd29a-reverse HindIII (AAGCTT) 25 Rd29a-forward KpnI (GGTACC) 26 Rd29bRd29b-reverse HindIII (AAGCTT) 27 Rd29b-forward KpnI (GGTACC) 28 Rab 18Rab18-reverse HindIII (AAGCTT) 29 Rab18-forward BglII (AGATCT) 30 Cor47Cor47-reverse HindIII (AAGCTT) 31 Cor47-forward BglII (AGATCT) 32

[0415] (1) Dreb2a Promoter

[0416] A cDNA encoding DRE (C-repeat) binding protein (DREB2A) has beenrecently identified (Liu et al. (1998) Plant Cell 10: 1391-1406). Thetranscription of the DREB2A gene is activated by dehydration andhigh-salt stress, but not by cold stress. The upstream untranslatedregion (166 bps) of dreb2a was used to BLAST-search the public database.A region containing the DREB2A promoter was identified in chromosome 5of Arabidopsis (Accession No. AB010692) between nucleotide positions51901-53955 (Table 6).

[0417] Two PCR primers designed to amplify the promoter region fromArabidopsis thaliana genomic DNA are as follows:

[0418] dreb2a-reverse: 5′-GCCCAAGCTTCAAGTTTAGTGAGCACTATGTGCTCG-3′ [SEQID NO: 19];

[0419] and dreb2a-forward: 5′-GGAAGATCTCCTTCCCAGAAACAACACAATCTAC-3′ [SEQID NO: 20].

[0420] The dre2ba-reverse primer includes a Hind III (AAGCTT)restriction site near the 5′-end of the primer and dreb2a-forward primerhas a Bgl II (AGATCT) restriction site at near 5′-end of the primer.These restriction sites may be used to facilitate cloning of thefragment into an expression vector.

[0421] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The reaction conditions that may be used in this PCR experiment are asfollows: Segment 1: 94° C., 2 minutes; Segment 2: 94° C., 30 seconds;60° C., 1 minute; 72° C., 3 minutes, for a total of 35 cycles; Segment3: 72° C. for 10 minutes. A PCR product of 2054 bp is expected.

[0422] The PCR products can be subject to electrophoresis in a 0.8%agarose gel and visualized by ethidium bromide staining. The DNAfragments containing the inducible promoter will be excised and purifiedusing a QIAQUICK gel extraction kit (Qiagen, Valencia, Calif.).

[0423] (2) P5CS Promoter

[0424] A cDNA for delta 1-pyrroline-5-carboxylate synthetase (P5CS) hasbeen isolated and characterized (Yoshiba et al. (1995) Plant J. 7:751-760). The cDNA encodes an enzyme involved in the biosynthesis ofproline under osmotic stress (drought/high salinity). The transcriptionof the P5CS gene was found to be induced by dehydration, high salt, andtreatment with plant hormone ABA, while it did not respond to heat orcold treatment.

[0425] A genomic DNA containing a promoter region of P5CS was identifiedby a BLAST search of GenBank using the upstream untranslated region (106bps) of the P5CS sequence (Accession No. D32138). The sequence for theP5CS promoter is located in the region between from nucleotide positions45472 to 47460 (Accession No. AC003000; Table 6).

[0426] Reverse and forward PCR primers designed to amplify this promoterregion from Arabidopsis thaliana genomic DNA are

[0427] P5CS-reverse primer 5′-GCCCAAGCTTGTTTCATTTTCTCCATGAAGGAGAT-3′[SEQ ID NO: 21]; and

[0428] P5CS-forward primer 5′-GGAAGATCTTATCGTCGTCGTCGTCTACCAAAACCACAC-3′[SEQ ID NO: 22].

[0429] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1988 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0430] (3) rd22 Promoter

[0431] A cDNA clone of rd22 was isolated from Arabidopsis underdehydration conditions (Yamaguchi-Shinozaki et al. (1993) Mol. Gen.Genet. 238: 17-25). Transcripts of rd22 were found to be induced by saltstress, water deficit and endogenous abscisic acid (ABA) but not by coldor heat stress. A promoter region was identified from GenBank by usingNucleotide Search WWW Entrez at the NCBI with the rd22 as a search word.The sequence for the rd22 promoter is located in the region betweennucleotide positions 17 to 1046 (Accession No. D10703; Table 6).

[0432] Reverse and forward PCR primers designed to amplify this promoterregion from Arabidopsis thaliana genomic DNA are

[0433] rd22-reverse primer 5′-GCTCTAAGCTTCACAAGGGGTTCGTTTGGTGC-3′ [SEQID NO: 23]; and

[0434] rd22-forward primer5′-GGGGTACCTTTTGGGAGTTGGAATAGAAATGGGTTTGATG-3′ [SEQ ID NO: 24].

[0435] The rd22-reverse primer includes a Hind III (AAGCTT) restrictionsite near the 5′-end of primer and rd22-forward primer has a KpnI(GGTACC) restriction site at near 5′-end of primer.

[0436] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1029 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0437] (4) rd29a Promoter

[0438] The rd29a and rb29b genes were isolated and characterized byShinozaki's group in Japan (Yamaguchi-Shinizaki et al. (1993) PlantPhysiol. 101: 1119-1120). Both rd29a and rb29b gene expressions werefound to be induced by desiccation, salt stress and exogenous ABAtreatment (Yamaguchi-Shinizaki et al. (1993) supra); Ishitani et al.(1998) Plant Cell 10: 1151-1161). The rd29a gene expression was inducedwithin 20 min after desiccation, but rd29b mRNA did not accumulate to adetectable level until 3 hours after desiccation. Expression of rd29acould also be induced by cold stress, whereas expression of rd29b couldnot be induced by low temperature.

[0439] A genomic clone carrying the rd29a promoter was identified byusing Nucleotide Search WWW Entrez at the NCBI with the rd29a as asearch word. The sequence for the rd29a promoter is located in theregion between nucleotide positions 3870 to 5511 (Accession No. D13044,Table 6).

[0440] Reverse and forward primers designed to amplify this promoterregion from Arabidopsis genomic DNA are:

[0441] rd29a-reverse primer 5′-GCCCAAGCTTAATTTTACTCAAAATGTTTTGGTTGC-3′[SEQ ID NO: 25]; and

[0442] rd29a-forward primer5′-CCGGTACCTTTCCAAAGATTTTTTTCTTTCCAATAGAAGTAATC-3′ [SEQ ID NO: 26].

[0443] The rd29a-reverse primer includes a Hind III (AAGCTT) restrictionsite near the 5′-end of primer and rd29a-forward primer has a KpnI(GGTACC) restriction site near 5′-end of primer.

[0444] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1641 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0445] (5) rd29b Promoter

[0446] A genomic clone carrying the rd29b promoter was identified byusing Nucleotide Search WWW Entrez at the NCBI with the rd29b as asearch word. The sequence for the rd29a promoter was located in theregion between nucleotide positions 90 to 1785 for rd29b (Accession No.D13044; Table 6).

[0447] Reverse and forward PCR primers designed to amplify this promoterregion from Arabidopsis thaliana genomic DNA are:

[0448] rd29b-reverse primer 5′-GCGGAAGCTTCATTTTCTGCTACAGAAGTG-3′ [SEQ IDNO: 27]; and

[0449] rd29b-forward primer5′-CCGGTACCTTTCCAAAGCTGTGTTTTCTCTTTTTCAAGTG-3′ [SEQ ID NO: 28].

[0450] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1695 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0451] (6) rab18 Promoter

[0452] A rab-related (responsive to ABA) gene, rab18 from Arabidopsishas been isolated. The gene encodes a hydrophilic, glycine-rich proteinwith the conserved serine- and lysine-rich domains. The rab18transcripts accumulate in plants exposed to water deficit or exogenousabscisic acid (ABA) treatment. A weak induction of rab18 mRNA by lowtemperature was also observed (Ishitani et al. (1998) Plant Cell 10:1151-1161).

[0453] A genomic DNA containing a promoter region of rab18 wasidentified by a BLAST search of GenBank using the upstream untranslatedregion (757 bps) of the rab18 sequence (Accession No. L04173). Thesequence of the rab18 promoter is located in the region betweennucleotide positions 8070 to 9757 (Accession No. AB013389).

[0454] Reverse and forward PCR primers designed and used to amplify thispromoter region from Arabidopsis thaliana genomic DNA are:

[0455] rab18-reverse primer5′-GCCCAAGCTTCAAATTCTGAATATTCACATATCAAAAAAGTG-3′ [SEQ ID NO: 29]; and

[0456] rab18-forward primer5′-GGAAGATCTGTTCTTCTTGTCTTAAGCAAACACTTTGAGC-3′ [SEQ ID NO: 30].

[0457] The rab18-reverse primer includes a Hind III (AAGCTT) restrictionsite near the 5′-end of the primer and rab18-forward primer has a Bgl II(AGATCT) restriction site near the 5′-end of the primer.

[0458] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1687 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0459] (7) Cor47 Promoter

[0460] The DNA sequence of cDNA for cold-regulated (cor47) gene ofArabidopsis thaliana was determined. Gilmour et al. (1992) Plant Molec.Biol. 18: 13-21). Expression of cor47 gene was induced by cold stress,dehydration and high NaCl treatment (Ishitani et al. (1998) Plant Cell.10: 1151-1161). The promoter region of cor47 gene was identified inGenBank by using Nucleotide Search WWW Entrez at the NCBI with the cor47as a search word. The sequence of the cor47 promoter is located in theregion between nucleotide positions 1-1370 (Accession No. AB004872;Table 6).

[0461] Reverse and forward PCR primers designed to amplify this promoterregion from Arabidopsis thaliana genomic DNA are:

[0462] cor47-reverse primer5′-GCCCAAGCTTTCGTCTGTTATCATACAAGGCACAAAACGAC-3′ [SEQ ID NO: 31]; and

[0463] cor47-forward primer5′-GGAAGATCTAGTTAATCTTGATTTGATTAAAAGTTTATATAG-3′ [SEQ ID NO: 32].

[0464] The cor47-reverse primer includes a Hind III (AAGCTT) restrictionsite near the 5′-end of the primer and cor47-forward primer has a Bgl II(AGATCT) restriction site near the 5′-end of the primer.

[0465] Total genomic DNA may be isolated from Arabidopsis thaliana(ecotype colombia) by using the CTAB method (Ausubel et al. (1992)Current Protocols in Molecular Biology (Greene & Wiley, New York)). Tennanograms of the genomic DNA can be used as a template in a PCR reactionunder conditions suggested by the manufacturer (Boehringer Mannheim).The PCR product is expected to be 1370 bps and may be PCR amplified andgel purified following the same protocol described for the dreb2apromoter.

[0466] Construction of the plasmids containing CBF1 and induciblepromoter. The expression binary vector pMEN020 contains a kanamycinresistance gene (neomycin phosphotransferase) for antibiotic selectionof the transgenic plants and a Spc/Str gene used for bacterial oragrobacterial selections. The pMEN020 plasmid is digested withrestriction enzymes such as HindIII and BglII to remove the 35Spromoter. The 35S promoter is then replaced with an inducible promoter.

[0467] (1) Cloning of the Inducible Promoter into pMEN020

[0468] The sequences of the inducible promoters that are PCR amplifiedand gel purified, as well as the plasmid pMEN020, are subject torestriction digestion with their respective restriction enzymes aslisted in Table 7. Both DNA samples are purified by using the Qiaquickpurification kit (Qiagen) and ligated at a ratio of 3:1 (vector toinsert). Ligation reactions using T4 DNA ligase (New England Biolabs,MA) are carried out at 16° C. for 16 hours. The ligated DNAs aretransformed into competent cells of the E. coli strain DH5α by using theheat shock method. The transformed cells are plated on LB platescontaining 100 μg/ml spectinomycin (Sigma-Aldrich, St. Louis, Mo.).Individual colonies are grown overnight in five milliliters of LB brothcontaining 100 μg/ml spectinomycin at 37° C.

[0469] Plasmid DNAs from transformants are purified by using QIAQUICKMini Prep kits (Qiagen) according to the manufacturer's instruction. Thepresence of the promoter insert is verified by restriction mapping withthe respective restriction enzymes as listed in Table 7 to cut out thecloned insert. The plasmid DNA is also subject to double-strand DNAsequencing analysis using a vector primer

[0470] E9.1 primer 5′-CAAACTCAGTAGGATTCTGGTGTGT-3′ [SEQ ID NO: 33].

[0471] (2) Cloning of the cbf1 Gene into the Plasmids Containing theInducible Promoters

[0472] To clone the CBF1 gene into the plasmids, different PCR primerswith suitable restriction sites for each plasmid are used to isolateCBF1 gene from Arabidopsis thaliana genomic DNA. The primers that may beused are listed in Table 8. TABLE 8 Promoter name Primer name Cloningsites Dreb2a Cbf1-reverse1 BglII (AGATCT) Cbf1-forward1 BamHI (GGATCC)P5CS Cbf1-reverse1 BglII (AGATCT) Cbf1-forward1 BamHI (GGATCC) Rd22Cbf1-reverse2 KpnI (GGTACC Cbf1-forward1 BamHI (GGATCC) Rd29aCbf1-reverse2 KpnI (GGTACC Cbf1-forward1 BamHI (GGATCC) Rd29bCbf1-reverse2 KpnI (GGTACC Cbf1-forward1 BamHI (GGATCC) Rab18Cbf1-reverse1 BglII (AGATCT) Cbf1-forward2 XbaI (TCTAGA Cor47Cbf1-reverse1 BglII (AGATCT) Cbf1-forward1 BamHI (GGATCC)

[0473] Two of the four available PCR primers (Table 8) are used forcloning the at-CBF1 gene into the expression vectors containing eachinducible promoter described above. The four primers have thesesequences:

[0474] cbf1-reverse 1 5′-GGAAGATCTTGAAACAGAGTACTCTGATCAATGAACTC-3′ [SEQID NO: 34],

[0475] cbf1-forward 1 5′-CGCGGATCCCTCGTTTCTACAACAATAAAATAAAATAAAATG-3′[SEQ ID NO: 35]

[0476] cbf1-reverse 2 5′-GGGGTACCTGAAACAGAGTACTCTGATCAATGAACTC-3′ [SEQID NO: 36], and

[0477] cbf1-forward 2 5′-GCTCTAGACTCGTTTCTACAACAATAAAATAAAATAAAATG-3′[SEQ ID NO: 37].

[0478] For example, for the Dreb2a, P5CS, and COR47 promoters that areligated to a BamHI and BglII flanked insert, the cbf1-reverse 1 andcbf1-forward 1 primers [SEQ ID NO: 34 and 35, respectively] are used toisolate CBF1 gene from Arabidopsis thaliana genomic DNA. Thecbf1-reverse primer includes a BglII (AGATCT) restriction site near the5′-end of the primer and cbf1-forward primer has a BamHI (GGATCC)restriction site near the 5′-end of the primer. A PCR product of 764 bpis expected. The genomic DNA (10 ng) is used as a template in a PCRreaction under conditions suggested by the manufacturer (BoehringerMannheim). The reaction conditions to be used in this PCR experiment areas follows: Segment 1: 94° C., 2 minutes; Segment 2: 94° C., 30 seconds;55° C., 1 minute; 72° C., 1 minute, for a total of 35 cycles; Segment 3:72° C. for 10 minutes.

[0479] The PCR products are subject to electrophoresis in a 0.8% agarosegel and visualized by ethidium bromide staining. The DNA fragmentcontaining cbf1 is excised and purified by using a Qiaquick gelextraction kit (Qiagen). The purified fragment and the vector pMBI2001containing the inducible promoter (Table 8) are each digested with BglIIand BamHI restriction enzymes at 37° C. for 2 hours. Both DNA samplesare purified by using the QIAQUICK purification kit (Qiagen) and ligatedat a ratio of 3:1 (vector to insert ratio). Ligation reactions using T4DNA ligase (New England Biolabs, MA) are carried out at 16° C. for 16hours. The ligated DNAs are transformed into competent cells of the E.coli strain DH5α by using the heat shock method. The transformation areplated on LB plates containing 100 (g/ml spectinomycin (Sigma-Aldrich).

[0480] Individual colonies are grown overnight in five milliliters of LBbroth containing 100 g/ml spectinomycin at 37° C. Plasmid DNA arepurified by using QIAQUICK Mini Prep kits (Qiagen). The presence of thecbf1 insert is verified by restriction mapping with BglII and BamHI. Theplasmid DNA is also subject to double-strand DNA sequencing analysis byusing vector primer E9.1

[0481] 5′-CAAACTCAGTAGGATTCTGGTGTGT-3′ [SEQ ID NO: 33].

[0482] The other primers shown in Table 8 and appropriate restrictionenzymes are used in a similar way to clone the CBF1 gene into plasmidscontaining the other inducible promoters. The resulting plasmids arelisted in Table 9 and shown in FIGS. 17A-17G.

[0483] A similar cloning strategy may be used to clone other genes, suchas cbf2, cbf3, and the other full length CBF genes listed in Table 9 andshown in FIG. 18 (new CBF gene table) into plasmids containing induciblepromoters. TABLE 9 Construct name Promoter name Figure name PMBI2008Dreb2a PMBI2009 P5CS PMBI2010 Rd22 PMBI2011 Rd29a PMBI2012 Rd29bPMBI2013 Rab18 PMBI2014 Cor47 FIG. 17G

Example 9

[0484] Transformation of Agrobacterium with Plasmids Containing CBF1Gene and Inducible Promoters

[0485] After the plasmid vectors containing CBF1 gene and induciblepromoters are constructed, these vectors may be used to transformAgrobacterium tumefaciens cells expressing the gene products. The stockof Agrobacterium tumefaciens cells for transformation may be made asdescribed by Nagel et al. (1990) FEMS Microbiol Letts 67: 325-328.Agrobacterium strain ABI may be grown in 250 ml LB medium(Sigma-Aldrich) overnight at 28° C. with shaking until an absorbance(A₆₀₀) of 0.5-1.0 is reached. Cells are harvested by centrifugation at4,000×g for 15 min at 4 C. Cells are then resuspended in 250 μl chilledbuffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells are centrifugedagain as described above and resuspended in 125 μl chilled buffer. Cellsare then centrifuged and resuspended two more times in the same HEPESbuffer as described above at a volume of 100 μl and 750 μl,respectively. Resuspended cells are then distributed into 40 μlaliquots, quickly frozen in liquid nitrogen, and stored at −80° C.

[0486] Agrobacterium cells are transformed with plasmids formed asdescribed above in Section 4B(2) following the protocol described byNagel et al. (1990) supra. For each DNA construct to be transformed,50-100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH8.0) is mixed with 40 μl of Agrobacterium cells. The DNA/cell mixture isthen transferred to a chilled cuvette with a 2 mm electrode gap andsubject to a 2.5 kV charge dissipated at 25 μF and 200 μF using a GenePulser II apparatus (Bio-Rad, Hercules Calif.). After electroporation,cells are immediately resuspended in 1.0 ml LB and allowed to recoverwithout antibiotic selection for 2-4 hours at 28° C. in a shakingincubator. After recovery, cells are plated onto selective medium of LBbroth containing 100 μg/ml spectinomycin (Sigma-Aldrich) and incubatedfor 24-48 h at 28° C. Single colonies are then picked and inoculated infresh medium. The presence of the plasmid construct are verified by PCRamplification and sequence analysis.

Example 10

[0487] Transformation of Arabidopsis Plants with Agrobacteriumtumefaciens Carrying Expression Vector for CBF1 Protein

[0488] After transformation of Agrobacterium tumefaciens with plasmidvectors containing CBF1 gene and inducible promoters, singleAgrobacterium colonies containing each of pMBI2008-pMBI2014 areidentified, propagated, and used to transform Arabidopsis plants.Briefly, 500 ml cultures of LB medium containing 100 ug/ml spectinomycinare inoculated with the colonies and grown at 28 C with shaking for 2days until an absorbance (A₆₀₀) of >2.0 is reached. Cells are thenharvested by centrifugation at 4,000×g for 10 min, and resuspended ininfiltration medium (½× Murashige and Skoog salts (Sigma-Aldrich), 1×Gamborg's B-5 vitamins (Sigma-Aldrich), 5.0% (w/v) sucrose(Sigma-Aldrich), 0.044 μM benzylamino purine (Sigma-Aldrich), 200 μl/LSilwet L-77 (Lehle Seeds) until an absorbance (A₆₀₀) of 0.8 is reached.

[0489] Prior to transformation, Arabidopsis thaliana seeds (ecotypeColumbia) are sown at a density of ˜10 plants per 4″ pot onto Pro-Mix BXpotting medium (Hummert International) covered with fiberglass mesh (18mm×16 mm). Plants are grown under continuous illumination (50-75μE/m²/sec) at 22-23 C with 65-70% relative humidity. After about 4weeks, primary inflorescence stems (bolts) are cut off to encouragegrowth of multiple secondary bolts. After flowering of the maturesecondary bolts, plants are prepared for transformation by removal ofall siliques and opened flowers.

[0490] The pots are then immersed upside down in the mixture ofAgrobacterium/infiltration medium as described above for 30 sec, andplaced on their sides to allow draining into a 1′×2′ flat surfacecovered with plastic wrap. After 24 h, the plastic wrap is removed andpots are turned upright. The immersion procedure is repeated one weeklater, for a total of two immersions per pot. Seeds are then collectedfrom each transformation pot and analyzed following the protocoldescribed below.

Example 11

[0491] Identification of Arabidopsis Primary Transformants

[0492] Seeds collected from the transformation pots are sterilizedessentially as follows. Seeds are dispersed into in a solutioncontaining 0.1% (v/v) Triton X-100 (Sigma-Aldrich) and sterile H₂O andwashed by shaking the suspension for 20 min. The wash solution is thendrained and replaced with fresh wash solution to wash the seeds for 20min with shaking. After removal of the second wash solution, a solutioncontaining 0.1% (v/v) Triton X-100 and 70% EtOH (Equistar) is added tothe seeds and the suspension is shaken for 5 min. After removal of theethanol/detergent solution, a solution containing 0.1% (v/v) TritonX-100 and 30% (v/v) bleach (Chlorox) is added to the seeds, and thesuspension is shaken for 10 min. After removal of the bleach/detergentsolution, seeds are then washed five times in sterile distilled H₂O. Theseeds are stored in the last wash water at 4° C. for 2 days in the darkbefore being plated onto antibiotic selection medium (1× Murashige andSkoog salts (pH adjusted to 5.7 with 1M KOH), 1× Gamborg's B-5 vitamins,0.9% phytagar (Life Technologies, Rockville, Md.), and 50 μg/Lkanamycin). Seeds are germinated under continuous illumination (50-75μE/m²/sec) at 22-23° C. After 7-10 days of growth under theseconditions, kanamycin resistant primary transformants (T₁ generation)are visible and are obtained for each of constructs pMBI2008-pMBI2014.These seedlings are transferred first to fresh selection plates wherethe seedlings continued to grow for 3-5 more days, and then to soil(Pro-Mix BX potting medium). Progeny seeds (T₂) are collected; kanamycinresistant seedlings selected and analyzed as described above.

Example 12

[0493] Transformation of Cereal Plants with Plasmid Vectors ContainingCBF1 Gene and Inducible Promoters.

[0494] Cereal plants, such as corn, wheat, rice, sorghum and barley, canalso be transformed with the plasmid vectors containing the CBF genesand inducible promoters to modify their biomass or increase theirtolerance to environmental stresses. In these cases, the cloning vector,pMEN020, is modified to replace the NptII coding region with the BARgene of Streptomyces hygroscopicus that confers resistance tophosphinothricin. The KpnI and BglII sites of the Bar gene are removedby site-directed mutagenesis with silent codon changes. After cloning ofthe inducible promoters into the modified plasmid by the same proceduresdescribed above, the at-cbf coding region of cbf1 gene is inserted intothe plasmid following the same procedures as described above. Theresulting plasmids are listed in Table 10. TABLE 10 Promoter nameConstruct name Dreb2a PMBI2015 P5CS PMBI2016 Rd22 PMBI2017 Rd29aPMBI2018 Rd29b PMBI2019 Rab18 PMBI2020 Cor47 PMBI2021

[0495] It is now routine to produce transgenic plants of most cerealcrops (Vasil (1994) Plant Molec. Biol. 25: 925-937) such as corn, wheat,rice, sorghum (Cassas et al. (1993) Proc. Natl. Acad Sci 90:11212-11216), and barley (Wan et al. (1994). Plant Physiol. 104: 37-48.Other direct DNA transfer methods such as the microprojectile gun orAgrobacterium tumefaciens-mediated transformation can be used for corn(Fromm et al. (1990) Bio/Technology 8: 833-839; Gordon-Kamm et al.(1990) Plant Cell 2: 603-618; Ishida (1990) Nature Biotechnology 14:745-750), wheat (Vasil et al. (1992) Bio/Technology 10: 667-674; Vasilet al. (1993) Bio/Technology 11: 1553-1558; Weeks et al. (1993) PlantPhysiol. 102: 1077-1084), rice (Christou (1991)Bio/Technology 9:957-962; Hiei et al. (1994) Plant J. 6: 271-282; Aldemita et al. (1996)Planta 199: 612-617; and Hiei et al. (1997) Plant Mol. Biol. 35:205-218). For most cereal plants, embryogenic cells derived fromimmature scutellum tissues are the preferred cellular targets fortransformation (Hiei et al. (1997) supra; Vasil (1994) Plant Molec.Biol. 25: 925-937).

[0496] Plasmids according to the present invention may be transformedinto corn embryogenic cells derived from immature scutellar tissue byusing microprojectile bombardment, with the A188XB73 genotype as thepreferred genotype (Fromm et al. (1990) Bio/Technology 8: 833-839;Gordon-Kamm et al. (1990) Plant Cell 2: 603-618). After microprojectilebombardment the tissues are selected on phosphinothricin to identify thetransgenic embryogenic cells (Gordon-Kamm et al. (1990) supra).Transgenic plants are regenerated by standard corn regenerationtechniques (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra).

[0497] The plasmids prepared as described above can also be used toproduce transgenic wheat and rice plants (Christou (1991) supra; Hiei etal. (1994) supra; Aldemita (1996) supra; Hiei et al. (1997) supra) byfollowing standard transformation protocols known to those skilled inthe art for rice and wheat Vasil, et al. (1992) supra; Vasil et al.(1993) supra; Weeks et al. (1993) supra), where the BAR gene is used asthe selectable marker.

Example 13

[0498] Transformation of Plants with Plasmid Vectors Containing CBF1Gene and Seed Specific Promoters.

[0499] The napin promoter from Brassica campestris (GenBank accessionno. M64632) is a seed specific promoter. A fragment of the napinpromoter (between nucleotides 1146 to 2148) is identified and isolatedby PCR amplification using a 5′ PCR primer containing a HindIII siteupstream of the promoter and a 3′ PCR primer containing a BamHI sitedownstream of the promoter. Deletion of the napin promoter to −211 and−152 have been shown to have reduced levels of expression (Ellerstrom etal. (1996) Plant Mol. Biol. 32: 1019-1027; Stålberg et al. (1996) Planta199: 515-519; Stålberg et al. (1993) Plant Mol. Biol. 23: 671-683).These 5′ deleted promoters are useful to have reduced levels of CBF1expression for applications where the larger napin promoter fragment istoo large.

[0500] Other seed-active promoters or deletions of these promoters canalso be isolated from genomic DNA by using the same method describedabove for the napin promoter. Examples of these promoters include butare not limited to the soybean 7S seed storage protein (Chen et al.(1989) Devel. Gen. 10: 112-122, the bean phaseolin promoter (cited inU.S. Pat. No. 5,003,045), the Arabidopsis 12S globulin (cruciferin)promoter (Pang et al. (1988) Plant Mol. Biol. 11: 805-820, the maizeglobulin1 promoter (Kriz et al. (1989) Plant Physiol. 91: 636; U.S. Pat.No. 5,773,691). These promoters maybe used for altering COR geneexpression in cereals such as corn, barley, wheat, rice and rye seeds.

[0501] The binary constructs containing seed-specific napin promoters(pMEN1001.1-4; pMEN1002.1-4; and pMEN1003.1-4) are used to transformcanola and rapeseed plants as described (Moloney et al. U.S. Pat. No.5,750,871), except that the Bar gene selectable marker is used.

[0502] These constructs are also used to transform regenerable barleycells by microprojectile bombardment (Wan et al. (1994) Plant Physiol.104: 37-48). After bombardment the tissues are selected onphosphinothricin by standard barley regeneration techniques (Wan andLemaux, supra).

Example 14

[0503] Identification of Homologous Sequence to CBF1 in Canola

[0504] This example describes the identification of homologous sequencesto CBF1 in canola using PCR. Degenerate primers were designed forregions of AP2 binding domain and outside of the AP2 (carboxyl terminaldomain). More specifically, the following degenerate PCR primers wereused:

[0505] Mol 368 (reverse) 5′-CAY CCN ATH TAY MGN GGN GT-3′

[0506] Mol 378 (forward) 5′-GGN ARN ARC ATN CCY TCN GCC-3′

[0507] Y: C/T, N: A/C/G/T, H: A/C/T, M: A/C, R: A/G)

[0508] Primer Mol 368 is in the AP2 binding domain of CBF1 (amino acidsequence: H P I Y R G V) while primer Mol 378 is outside the AP2 domain(carboxyl terminal domain)(amino acid sequence: M A E G M L L P).

[0509] The genomic DNA isolated from Brassica Napus was PCR amplified byusing these primers following these conditions: an initial denaturationstep of 2 min at 93° C.; 35 cycles of 93° C. for 1 min, 55° C. for 1min, and 72° C. for 1 min; and a final incubation of 7 min at 72° C. atthe end of cycling.

[0510] The PCR products were separated by electrophoresis on a 1.2%agarose gel and, transferred to nylon membrane and hybridized with theAT CBF1 probe prepared from Arabidopsis genomic DNA by PCRamplification. The hybridized products were visualized by colorimetricdetection system (Boehringer Mannheim) and the corresponding bands froma similar agarose gel were isolated (By Qiagen Extraction Kit). The DNAfragments were ligated into the TA clone vector from TOPO TA Cloning Kit(Invitrogen) and transformed into E. coli strain TOP10 (Invitrogen).

[0511] Seven colonies were picked and the inserts were sequenced on anABI 377 machine from both strands of sense and antisense after plasmidDNA isolation. The DNA sequence was edited by sequencer and aligned withthe AtCBF1 by GCG software and NCBI blast searching.

[0512]FIG. 16 shows an amino acid sequence of a homolog (CAN1; SEQ IDNO: 17) identified by this process and its alignment to the amino acidsequence of CBF1. The nucleic acid sequence for CAN1 is listed herein asSEQ ID NO: 18.

[0513] As illustrated in FIG. 16, the amino acid sequence alignment infour regions of BN-CBF1 shows 82% identity in the AP2 binding domainregion and range from 75% to 83% with some alignment gaps due to regionsof lesser homology or introns in the genomic sequence. The aligned aminoacid sequences show that the polypeptide encoded by the BNCBF1 gene has88% identity in the AP2 domain region and 85% identity outside the AP2domain when aligned for two insertion sequences that are outside the AP2domain. The extra amino acids in the two insertion regions are eitherdue to the presence of introns in this region of the BNCBF1 gene, as itwas derived from genomic DNA, or could be due to extra amino acids inthese regions of the BNCBF1 gene. Isolation and sequencing of a cDNA ofthe BNCBF1 gene using the genomic DNA as a probe will resolve this.

Example 15

[0514] Identification of Homologous Sequence to CBF1 in Canola and OtherSpecies

[0515] A PCR strategy similar to that described in Example 14 was usedto isolate additional CBF homologs from Brassica juncea, Brassica napus,Brassica oleracea, Brassica rapa, Glycine max, Raphanus sativus and Zeamays. The nucleotide (for example, bjCBF1) and peptide sequences (forexample, BJCBF1-PEP) of these isolated CBF homologs are shown in FIGS.18A and 18B, respectively. Table 11 lists the sequence names and SEQ IDNOs of these isolated CBF homologs. The PCR primers are internal to thegene so partial gene sequences are initially obtained. The full lengthsequences of some of these genes were further isolated by inverse PCR orligated linker PCR. One skilled in the art can use the conserved regionsin these genes to design PCR primers to isolate additional CBF genes.TABLE 11 SEQ % DNA SEQ Name ID NO: Peptide SEQ Name SEQ ID NO: ID*bjCBF1 38 BJCBF1-PEP 39 87 bjCBF2 40 BJCBF2-PEP 41 85 bjCBF3 42BJCBF3-PEP 43 85 bjCBF4 44 BJCBF4-PEP 45 93 bnCBF1 46 BNCBF1-PEP 47 88bnCBF2 48 BNCBF2-PEP 49 87 bnCBF3 50 BNCBF3-PEP 51 87 bnCBF4 52BNCBF4-PEP 53 88 bnCBF5 54 BNCBF5-PEP 55 88 bnCBF6 56 BNCBF6-PEP 57 88bnCBF7 58 BNCBF7-PEP 59 87 bnCBF8 60 BNCBF8-PEP 61 88 bnCBF9 62BNCBF9-PEP 63 85 boCBF1 64 BOCBF1-PEP 65 88 boCBF2 66 BOCBF2-PEP 67 87boCBF3 68 BOCBF3-PEP 69 88 boCBF4 70 BOCBF4-PEP 71 88 boCBF5 72BOCBF5-PEP 73 87 brCBF1 74 BRCBF1-PEP 75 88 brCBF2 76 BRCBF2-PEP 77 88brCBF3 78 BRCBF3-PEP 79 88 brCBF4 80 BRCBF4-PEP 81 88 brCBF5 82BRCBF5-PEP 83 88 brCBF6 84 BRCBF6-PEP 85 88 brCBF7 86 BRCBF7-PEP 87 88gmCBF1 88 GMCBF1-PEP 89 87 rsCBF1 90 RSCBF1-PEP 91 88 rsCBF2 92RSCBF2-PEP 93 88 zmCBF1 94 ZMCBF1-PEP 95 80

[0516]FIG. 19A shows an amino acid alignment of the AP2 domains of theCBF proteins listed in Table 11 with their consensus sequenceshighlighted. FIG. 19A also provides a comparison of the consensussequence with that of the tobacco DNA binding protein EREBP2 (SEQ ID NO:168; Okme-Takagi et al. (1995) supra). The AP2 domain sequences of theseCBF proteins are: atcbf2 (SEQ ID NO:137); atcbf3 (SEQ ID NO: 138);atcbf1 (SEQ ID NO: 139); bjcbf4 (SEQ ID NO: 140); bocbf2 (SEQ ID NO:141); rscbf2 (SEQ ID NO: 142); bjcbf2 (SEQ ID NO: 143); bncbf7 (SEQ IDNO: 144); bjcbf3 (SEQ ID NO: 145); bncbf2 (SEQ ID NO: 146); bncbf3 (SEQID NO: 147); bncbf1 (SEQ ID NO: 148); bncbf6 (SEQ ID NO: 149); bncbf8(SEQ ID NO: 150); bocbf3 (SEQ ID NO: 151); brcbf2 (SEQ ID NO: 152);bncbf4 (SEQ ID NO: 153); bncbf5 (SEQ ID NO: 154); bocbf4 (SEQ ID NO:155); brcbf1 (SEQ ID NO: 156); brcbf3 (SEQ ID NO: 157); brcbf4 (SEQ IDNO: 158); brcbf5 (SEQ ID NO: 159); brcbf6 (SEQ ID NO: 160); brcbf7 (SEQID NO: 161); rscbf1 (SEQ ID NO: 162); bocbf1 (SEQ ID NO: 163); bocbf5(SEQ ID NO: 164); bncbf9 (SEQ ID NO: 165); zmcbf1 (SEQ ID NO: 166); andgmcbfl (SEQ ID NO: 167).

[0517] As can be seen from the consensus sequence shown in FIG. 19A, asignificant portion of the AP2 domain is conserved among the differentCBF proteins. In view of this data, Applicants use the conservedsequence in the AP2 domain to define a class of AP2 domain proteinscomprising this conserved sequence.

[0518]FIG. 19B shows an amino acid alignment of the AP2 domains shown inFIG. 19A and the AP2 doamins of dreb2a (SEQ ID NO:169) and dreb2b (SEQID NO: 170) and a consensus sequence between the proteins highlighted.As can be seen, a very high degree of homology exists between AP2domains shown in FIG. 19A and dreb2a and dreb2b. Applicants employ theconserved sequence in the AP2 domain shown in FIG. 19B to define abroader class of AP2 domain proteins that are capable of binding to CCGregulatory region.

[0519]FIG. 19C shows an amino acid alignment of the AP2 domains shown inFIG. 19B and the AP2 domain of tiny (SEQ ID NO: 171) and a consensussequence between the proteins highlighted. As can be seen, a very highdegree of homology exists between AP2 domains shown in FIG. 19A, dreb2a(SEQ ID NO: 169), dreb2b (SEQ ID NO:170), and tiny (SEQ ID NO:171).Applicants employ the conserved sequence in the AP2 domain shown in FIG.19C to define a yet broader class of AP2 domain proteins that arecapable of binding to CCG regulatory region.

[0520]FIG. 19D shows a consensus sequence corresponding to thedifference between the consensus sequence shown in FIG. 19A and tiny.Applicants employ the highlighted portion of the conserved sequenceshown in FIG. 19D to define a group of amino acid residues that may becritical to binding to a CCG regulatory region.

[0521]FIG. 19E shows a consensus sequence corresponding to thedifference between the consensus sequence shown in FIG. 19B and tiny.Applicants employ the highlighted portion of the conserved sequenceshown in FIG. 19E to define another group of amino acid residues thatmay be critical to binding to a CCG regulatory region.

[0522]FIG. 20 shows the amino acid alignment of the amino terminus ofthe CBF proteins with their consensus sequence highlighted. Thesequences of these CBF proteins are: brcbf3 (SEQ ID NO: 172); brcbf6(SEQ ID NO: 173); bncbf5 (SEQ ID NO: 174); atcbf2 (SEQ ID NO: 175);atcbf3 (SEQ ID NO: 176); atcbf1 (SEQ ID NO: 177); bncbf2 (SEQ ID NO:178); bncbf6 (SEQ ID NO: 179); bocbf3 (SEQ ID NO: 180); bncbf3 (SEQ IDNO: 181); bncbf8 (SEQ ID NO: 182); bncbf9 (SEQ ID NO: 183); brcbf2 (SEQID NO: 184); bocbf5 (SEQ ID NO: 185); bocbf2 (SEQ ID NO: 186); rscbf2(SEQ ID NO: 187); bncbf4 (SEQ ID NO: 188); bncbf7 (SEQ ID NO: 189);bocbf4 (SEQ ID NO: 190); brcbf7 (SEQ ID NO: 191); brcbf4 (SEQ ID NO:192); brcbf5 (SEQ ID NO: 193); and rscbf1 (SEQ ID NO: 194).

[0523] As can be seen from the consensus sequence shown in FIG. 20, asignificant portion of the amino terminus of CBF proteins is conservedamong the different CBF proteins. In view of this data, Applicantsemploy the conserved sequence in the amino terminus domain to define aclass of proteins comprising this conserved sequence. Of note, theconserved sequence corresponding to amino acids 31-37 of SEQ ID NO: 2,PKKPAGR; SEQ ID NO: 326), amino acids 35-40 of SEQ ID NO: 2 (AGRKKF; SEQID NO: 325), or amino acids 42-46 of SEQ ID NO: 2 (ETRHP; SEQ ID NO:321), can define the class of CBF proteins. In addition, the consensussequence corresponding to amino acids 31-37 of SEQ ID NO: 2 PKXXAGR; SEQID NO: 319), amino acids 35-40 of SEQ ID NO: 2 (AGRXKF; SEQ ID NO: 320),or amino acids 42-46 of SEQ ID NO: 2 (ETRHP; SEQ ID NO: 321), wherein Xis any amino acid residue, also can define the class of CBF proteins.

[0524]FIG. 21A shows the amino acid alignment of the carboxy terminus of24 CBF proteins with their consensus sequences highlighted. Thesequences of these CBF proteins are: bncbf3 (SEQ ID NO: 195; SEQ ID NO:219; SEQ ID NO: 243); bncbf9 (SEQ ID NO: 196; SEQ ID NO: 220; SEQ ID NO:244); brcbf2 (SEQ ID NO: 197; SEQ ID NO: 221; SEQ ID NO: 245); bncbf1(SEQ ID NO: 198; SEQ ID NO: 222; SEQ ID NO: 246); bncbf8 (SEQ ID NO:199; SEQ ID NO: 223; SEQ ID NO: 247); bncbf6 (SEQ ID NO: 200; SEQ ID NO:224; SEQ ID NO: 248); bocbf3 (SEQ ID NO: 201; SEQ ID NO: 225; SEQ ID NO:249); bncbf2 (SEQ ID NO: 202; SEQ ID NO: 226; SEQ ID NO: 250); bocbf5(SEQ ID NO: 203; SEQ ID NO: 227; SEQ ID NO: 251); brcbf5 (SEQ ID NO:204; SEQ ID NO: 228; SEQ ID NO: 252); rscbf1 (SEQ ID NO: 205; SEQ ID NO:229; SEQ ID NO: 253); bncbf4 (SEQ ID NO: 206; SEQ ID NO: 230; SEQ ID NO:254); bocbf4 (SEQ ID NO: 207; SEQ ID NO: 231; SEQ ID NO: 255); bncbf5(SEQ ID NO: 208; SEQ ID NO: 232; SEQ ID NO: 256); brcbf7 (SEQ ID NO:209; SEQ ID NO: 233; SEQ ID NO: 257); brcbf6 (SEQ ID NO: 210; SEQ ID NO:234; SEQ ID NO: 258); bocbf1 (SEQ ID NO: 211; SEQ ID NO: 235; SEQ ID NO:259); bjcbf2 (SEQ ID NO: 212; SEQ ID NO: 236; SEQ ID NO: 260); bjcbf3(SEQ ID NO: 213; SEQ ID NO: 237; SEQ ID NO: 261); bncbf7 (SEQ ID NO:214; SEQ ID NO: 238; SEQ ID NO: 262); rscbf2 (SEQ ID NO: 215; SEQ ID NO:239; SEQ ID NO: 263); atcbf1 (SEQ ID NO: 216; SEQ ID NO: 240; SEQ ID NO:264); atcbf2 (SEQ ID NO:217; SEQ ID NO: 241; SEQ ID NO: 265); and atcbf3(SEQ ID NO: 218; SEQ ID NO: 242; SEQ ID NO: 266).

[0525] As can be seen from the consensus sequence shown in FIG. 21A, asignificant portion of the carboxy terminus of CBF proteins is conservedamong the different CBF proteins. In view of this data, Applicantsemploy the conserved sequence in the carboxy terminus domain to define aclass of proteins comprising this conserved sequence.

[0526]FIG. 21B shows the amino acid alignment of the carboxy terminus of9 CBF proteins with their consensus sequences highlighted. The sequencesof these CBF proteins are: bncbf3 (SEQ ID NO: 267; SEQ ID NO: 276; SEQID NO: 285); bncbf9 (SEQ ID NO: 268; SEQ ID NO: 277; SEQ ID NO: 286);brcbf2 (SEQ ID NO: 269; SEQ ID NO: 278; SEQ ID NO: 287); bncbfl (SEQ IDNO: 270; SEQ ID NO: 279; SEQ ID NO: 288); bncbf8 (SEQ ID NO: 272; SEQ IDNO: 280; SEQ ID NO: 289); bncbf6 (SEQ ID NO: 273; SEQ ID NO: 281; SEQ IDNO: 290); bocbf3 (SEQ ID NO: 274; SEQ ID NO: 282; SEQ ID NO: 291);bncbf2 (SEQ ID NO: 274; SEQ ID NO: 283; SEQ ID NO: 292); bocbf5 (SEQ IDNO: 275; SEQ ID NO: 284; SEQ ID NO: 293).

[0527] As can be seen from the consensus sequence shown in FIG. 21B, agreater portion of the carboxy terminus is conserved when these nine CBFproteins are used. In view of this data, Applicants employ the conservedsequence in the carboxy terminus domain to define another class ofproteins comprising this conserved sequence.

Example 16

[0528] Homologous CBF Encoding Genes in other Plants.

[0529] This example shows that homologous sequences to CBF1 are presentin other plants. The presence of these homologous sequences suggest thatthe same or similar cold regulated environmental stress responseregulatory elements such as the C-repeat/DRE of Arabidopsis (CCGAC)exist in other plants. This example serves to indicate that genes withsignificant homology to CBF1, CBF2 and CBF3 exist in a wide range ofplant species.

[0530] Total plant DNAs from Arabidopsis thaliana, Nicotiana tabacum,Lycopersicon pimpinellifolium, Prunus avium, Prunus cerasus, Cucumissativus, and Oryza sativa were isolated according to Stockinger al(Stockinger et al. (1996) J. Heredity 87: 214-218). Approximately 2 to10 μg of each DNA sample was restriction digested, transferred to nylonmembrane (Micron Separations, Westboro Mass.) and hybridized accordingto Walling et al. (Walling et al. (1988) Nucleic Acids Res. 16:10477-10492). Hybridization conditions were: 42° C. in 50% formamide,5×SSC, 20 mM phosphate buffer 1× Denhardt's, 10% dextran sulfate, and100 μg/ml herring sperm DNA. Four low stringency washes at RT in 2×SSC,0.05% Na sarcosyl and 0.02% Na₄ pyrophosphate were performed prior tohigh stringency washes at 55° C. in 0.2×SSC, 0.05% Na sarcosyl and 0.01%Na₄ pyrophosphate. High stringency washes were performed until no countswere detected in the washout. The BclI-BglII fragment of CBF1(Stockinger et al. (1997) Proc Natl Acad Sci 94: 1035-1040) was gelisolated (Sambrook et al. (1989), supra) and direct prime labeled(Feinberg et al. (1982) Anal. Biochem. 132: 6-13) using the primer MT117

[0531] (TTGGCGGCTACGAATCCC; SEQ ID NO: 16).

[0532] Specific activity of the radiolabelled fragment was approximately4×10⁸ cpm/μg. Autoradiography was performed using HYPERFILM-MP(Amersham) at −80° C. with one intensifying screen for 15 hours.

[0533] Autoradiography of the gel showed that DNA sequences fromArabidopsis thaliana, Nicotiana tabacum, Lycopersicon pimpinellifolium,Prunus avium, Prunus cerasus, Cucumis sativus, and Oryza sativahybridized to the labeled BclI, BglII fragment of CBF1. These resultssuggest that homologous CBF encoding genes are present in a variety ofother plants.

Example 17

[0534] Identification of CBF1 Homologs CBF2 and CBF3 Using CBF1

[0535] This example describes two homologs of CBF1 from Arabidopsisthaliana and named them CBF2 and CBF3.

[0536] CBF2 and CBF3 have been cloned and sequenced as described below.The sequences of the DNA and encoded proteins are set forth in SEQ IDNOs: 12 and 13, 14 and 15, and FIGS. 12 and 13.

[0537] A lambda cDNA library prepared from RNA isolated from Arabidopsisthaliana ecotype Columbia (Lin et al. (1992) Plant Physiol. 99: 519-525)was screened for recombinant clones that carried inserts related to theCBF1 gene (Stockingeret al. (1997) Proc Natl Acad Sci 94: 1035-1040).CBF1 was ³²P-radiolabeled by random priming (Sambrook et al. (1989)supra) and used to screen the library by the plaque-lift technique usingstandard stringent hybridization and wash conditions (Hajela et al.(1990) Plant Physiol.93: 1246-1252; Sambrook et al. (1989), supra;6×SSPE buffer, 60° C. for hybridization and 0.1×SSPE buffer and 60° C.for washes). Twelve positively hybridizing clones were obtained and theDNA sequences of the cDNA inserts were determined at the MSU-DOE PlantResearch Laboratory sequencing facility. The results indicated that theclones fell into three classes. One class carried inserts correspondingto CBF1. The two other classes carried sequences corresponding to twodifferent homologs of CBF1, designated CBF2 and CBF3. The nucleic acidsequences and predicted protein coding sequences for CBF1 (SEQ ID NO:1and SEQ ID NO:2, respectively), CBF2 (SEQ ID NO:12 and SEQ ID NO:13,respectively), and CBF3 (SEQ ID NO:14 and SEQ ID NO:15, respectively)appear in the Sequence Listing.

[0538] A comparison of the nucleic acid sequences of CBF1, CBF2 and CBF3indicate that they are 83 to 85% identical as shown in Table 12. FIG. 14shows the amino acid alignment of proteins CBF1, (SEQ ID NO:2), CBF2(SEQ ID NO:13), and CBF3 (SEQ ID NO:15). TABLE 12 Percent identity^(a)DNA^(b) Polypeptide cbf1/cbf2 85 86 cbf1/cbf3 83 84 cbf2/cbf3 84 85

[0539] Similarly, the amino acid sequences of the three CBF polypeptidesrange from 84 to 86% identity. An alignment of the three amino acidicsequences reveals that most of the differences in amino acid sequenceoccur in the acidic C-terminal half of the polypeptide. This region ofCBF1 serves as an activation domain in both yeast and Arabidopsis (notshown).

[0540] Residues 47 to 106 of CBF1 correspond to the AP2 domain of theprotein, a DNA binding motif that, to date, has only been found in plantproteins. A comparison of the AP2 domains of CBF1, CBF2, and CBF3indicates that there are a few differences in amino acid sequence. Thesedifferences in amino acid sequence might have an effect on DNA bindingspecificity.

Example 18

[0541] Activation of Transcription in Yeast Containing C-Repeat/DREUsing CBF1, CBF2, and CBF3

[0542] This example shows that CBF1, CBF2, and CBF3 activatetranscription in yeast containing CRT/DREs upstream of a reporter gene.The CBFs were expressed in yeast under control of the ADC1 promoter on a2μ plasmid (pDB20.1; Berger et al. (1992) Cell 70: 251-265). Constructsexpressing the different CBFs were transformed into yeast reporterstrains that had the indicated CRT/DRE upstream of the lacZ reportergene. Copy number of the CRT/DREs and its orientation relative to thedirection of transcription from each promoter is indicated by thedirection of the arrow.

[0543]FIG. 15 is a graph showing transcription regulation of CRT/DREcontaining reporter genes by CBF1, CBF2, and CBF3 genes in yeast. InFIG. 15, the vertical lines across the arrows of the COR15a constructrepresent the m3cor15a mutant CRT/DRE construct. Each CRT/DRE-lacZconstruct was integrated into the URA3 locus of yeast. Error barsrepresent the standard deviation derived from three replicatetransformation events with the same CBF activator construct into therespective reporter strain. Quantitative B-gal assays were performed asdescribed by Rose and Botstein (Rose et al. (1983) Methods Enzymol. 101:167-180).

Example 19

[0544] Identification and Isolation of Novel CBF-Related Polypeptides

[0545] Additionally, we identified novel CBF-related polypeptides fromsoybean, wheat, rice, and rye plants.

[0546] Soybean seeds were bought from a local supermarket (packaged byJAMECO Co, San Francisco, Calif.). DNA and mRNA were isolated usingstandard procedures (Ausubel et al. (1998) Current Protocols inMolecular Biology (Greene & Wiley, New York)). A soybean seedling cDNAlibrary was also constructed using standard procedures. Based on thesequence of the Arabidopsis CBF1 gene (SEQ ID NO: 1), degenerate primers

[0547] O368 (CAYCCNATHTAYMGNGGNGT (SEQ ID NO: 104));

[0548] O376 (GCNGCYTCNGCNGCNGCYTTYTGDAT (SEQ ID NO: 105)); and

[0549] O2953 (AARAARTTYMGNGARACNMGNCAY (SEQ ID NO: 106))

[0550] were designed. O376 and O2953 were first used in a PCR experimentusing soybean genomic DNA as template. The product from this reactionwas excised from the gel, purified (Ausubel et al. (1998) supra), andused as a template in a second round of PCR using primers O368 and O376.Then, the PCR product was cloned into pGEM-T and sequenced using T7 andsp6 primers (Promega Corp).

[0551] Based on the sequence of the cloned soybean clone, 3′ rapidamplification of cDNA ends (RACE) was performed using the MARATHON cDNAamplification kit (Clontech, Palo Alto, Calif.). Generally, the methodentailed first isolating poly(A) mRNA, synthesize first and secondstrand cDNA to generate double stranded cDNA, blunting cDNA ends,followed by ligation of the Marathon™ Adaptor to the cDNA to form alibrary of adaptor-ligated double-stranded cDNA. Gene-specific primerswere designed to be used along with adaptor specific primers for 3′ RACEreactions. Often, nested primers were used to increase PCR specificity.In this case, the 3′ nested primers were

[0552] O5436 (GGAGGAACACGGATAAGTGGGTAAG (SEQ ID NO: 107)) and

[0553] O5437 (AGGATTTGGCTGGGGACTTTTCC (SEQ ID NO: 108)).

[0554] The resulting RACE fragment was cloned into the pGEM-T vector(Promega Corp) and sequenced using T7 and sp6 primers. The cloned insertwas then labeled using the DIG DNA Labeling and Detection Kit followingthe manufacture's instructions (Boehringer Mannheim), and the labeledprobe was used to screen the soybean cDNA library using standardprocedures and hybridization conditions (Ausubel et al. (1998) supra).SEQ ID NO: 127 was isolated in this manner.

[0555] Rice seeds were obtained from the laboratory of Dr. Pam Ronald atUC Davis. Corn, wheat, and rye seeds were obtained from the USDA, ARSNational Small Grains Research Facility, Aberdeen, Id. DNA and mRNA wereisolated using standard procedures. Seedling cDNA libraries were alsoconstructed using standard procedures.

[0556] In order to isolate CBF1 homologs from monocotyledon species,CBF1 polypeptide sequence was used to identify related sequences frompublic plant sequence databases. The tblastn sequence analysis programwas employed. A rice homolog (Ace. No. AB023482) was identified ashaving a P value of 6.3e-17. Based on its sequence, primers

[0557] O18016 (ACGCGTCGACCCATCATCACCGAGATCGACTCGAC (SEQ ID NO: 109)) and

[0558] O18017 (ATAAGAATGCGGCCGCTCATTGTTCGCTCACTGGGAG (SEQ ID NO: 110)

[0559] were synthesized, and the rice gene was isolated from ricegenomic DNA by a standard PCR procedure using those primers. Theamplified fragment was cloned into the pGEM-T vector following themanufacture's protocol (Promega Corp). The clone was sequenced usingO18016, O18017,

[0560] O18035 (GCTGACAGAACGGGTGCCGA (SEQ ID NO: 111)) and

[0561] O18036 (TGACCGTTTCTGGATAGGCA (SEQ ID NO: 112)).

[0562] Based on the rice sequence, primers

[0563] O18065 (GGCCGGCGGGGCGAACCAAGTTCC (SEQ ID NO: 113)) and

[0564] O18066 (AGGCAGAGTCGGCGAAGTTGAGGC (SEQ ID NO: 114))

[0565] were synthesized. These primers were used to isolate rye andwheat CBF gene fragments by PCR from their respective cDNA libraries.For some of the PCR reactions outlined above, a PCR optimization kit(Boehringer Mannheim) was used. The PCR product was cloned into thepGEM-T vector (Promega Corp). To isolate full-length rye cDNAs, the ryefragment was then labeled with ³²P dCTP using the High Prime DNALabeling Kit (Boehringer Mannheim). Purified radiolabeled probes wereused to probe a rye cDNA library using standard conditions (see Ausubelet al. Current Protocols in Molecular Biology, supra section 6.3). SEQID NO: 115, 117, 119, 121 and 123, which are rye CBF-related sequences,were isolated in this manner. SEQ ID NO: 125 is a CBF-related peptidesequence identified from wheat.

[0566] The percent sequence identity of the AP2 regions of polypeptideSEQ ID NO: 116, 118, 120, 122, 124 and 126, with CBF1 (SEQ ID NO: 2),are shown in Table 13. The percent sequence identity between thedifferent polypeptides provided in the Sequence Listing varied from 53%to 96% over most of the length of the sequences. Generally, thesesequences comprise an AP2 domain comprising amino acids 45, 46, 48,50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91,93-96, 99, 101, 102 and 104-106 of SEQ ID NO: 2 or comprise one or moreof the following peptides: PKXXAGR (SEQ ID NO:319; amino acids 31-37 ofSEQ ID NO: 2), or AGRXKF (SEQ ID NO: 320; amino acids 35-40 of SEQ IDNO: 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO: 2).TABLE 13 Peptide % DNA SEQ Name SEQ ID NO: SEQ Name SEQ ID NO: ID* RyeCBF20 115 Rye CBF20 116 69 PEP Rye CBF28 117 Rye CBF28- 118 70 PEP RyeCBF46 119 Rye CBF46- 120 71 PEP Rye CBF7 121 Rye CBF7- 122 67 PEP RyeCBF71 123 Rye CBF71- 124 71 PEP Wheat CBF 125 Wheat CBF- 126 71 PEP

Example 20

[0567] Identification and Isolation of Additional CBF Orthologs fromother Plants

[0568]Medicago truncatula

[0569] Orthologous CBF sequences from Medicago truncatula wereidentified by BLAST analysis (TBLASTN 2.1.3 [Apr. 1, 2001]). Medicagotruncatula ESTs in the Genbank public database were queried using theconserved domain from Arabidopsis CBF1, CBF2, and CBF3. The Arabidopsissequences included the entire AP2/EREBP conserved domain flanked by theCBF signature sequences PKK/RPAGRxKFxETRHP and DSAWR of ArabidopsisCBF1, CBF2, CBF3, and CBF4 (SEQ ID NOs: 327, 328, 329, and 330,respectively). EST sequences were considered positive hits if they hadan HSP BLAST score of at least 100 bits over at least 95% of the querysequence.

[0570] All positive EST sequences were organized into contigs using theSEQUENCHER assembly program (SEQUENCHER version 3.1, Gene CodesCorporation, Ann Arbor Mich.). At least seven separate sequences wereidentified (SEQ ID NOs: 294, 295, 296, 297, 298, 299, and 300). Thosethat had complete predicted coding regions with a start and a stop codonwere cloned directly from cDNA or genomic DNA by PCR using primers inthe 5′ and 3′ flanking regions. RACE (SMART RACE, BD Clontech) was usedto identify the start and stop codons of genes that did not havefull-length coding sequences available in the public database. Thesesequences were then cloned as above. All sequences (SEQ ID NOs: 294,295, 296, 297, 298, 299, and 300) were cloned into the transformationvector pMEN65, or a modified version with GATEWAY compatible sites(GATEWAY Technology, Invitrogen Life Technologies, Carlsbad Calif.).

[0571]Oryza sativa

[0572] Rice orthologs were identified in the GenBank public database asdescribed above for Medicago truncatula. Positive hits were organizedinto contigs using Sequencher. Ten full length sequences (SEQ ID NOs:301, 302, 303, 304, 305, 306, 307, 308, 309, and 310) were identifiedand cloned from genomic DNA by PCR using primers in the 5′ and 3′untranslated regions. All genes were cloned into the transformationvector pMEN65, or a modified version with GATEWAY compatible sites.

[0573]Medicago saliva

[0574] No putative alfalfa orthologs were identified in GenBank publicdatabase using BLAST analysis as described above for M. truncatula.However, because M. truncatula and M. sativa species are so closelyrelated, three alfalfa CBF orthologs (SEQ ID NOs: 316, 317, and 318)were cloned directly from alfalfa genomic DNA using the M. truncatulacloning primers. All genes were cloned into the transformation vectorpMEN65.

[0575]Zea mays

[0576] Maize orthologs were identified using BLAST analysis as describedfor M. truncatula in a proprietary cDNA database. Five orthologs (SEQ IDNOs: 311, 312, 313, 314, and 315) were cloned with PCR primers in the 5′and 3′ untranslated regions and inserted into pMEN65.

[0577] Alignment with Arabidopsis CBFs

[0578] Polynucleotides isolated from Medicago truncatula (SEQ ID NOs:294-300) which encoded CBF-related peptide sequences are shown comparedwith Arabidopsis CBF1, CBF2, and CBF3 in FIG. 39 (SEQ ID NOs: 2, 15, 13,and 97, respectively). Polynucleotides isolated from Oryza sativa (SEQID NOs: 301-310) which encoded CBF-related peptide sequences are showncompared with Arabidopsis CBF1, CBF2, and CBF3 in FIG. 40 (SEQ ID NOs:2, 15, 13, and 97, respectively). Polynucleotides isolated from Zea mays(SEQ ID NOs: 311-315) which encoded CBF-related peptide sequences areshown compared with Arabidopsis CBF1, CBF2, CBF3, and CBF4 in FIG. 41(SEQ ID NOs: 2, 15, 13, and 97, respectively).

Example 21

[0579] Testing of CBF Orthologs in a Transient Assay

[0580] Preparation of Transformant Vectors

[0581] Seven clones from M. truncatula, ten clones from rice, and threeclones from alfalfa (M. sativa) were tested for CBF activity in atransient assay. CBF activity was determined by activation of the RD29apromoter, quantitated by GUS activity. Results are shown on FIG. 42. Inaddition, rice CBF G3376 (SEQ ID NO:307) activated transcription fromthe RD29a promoter by greater than four-old when compared with theplasmid pMEN 65 vector control.

[0582] CBF clones were introduced into the pMEN 65 vector under thetranscriptional control of the 35S promoter to create a 35S::CBF clone.

[0583] 35S::CBF clones transformed into Agrobacterium cells were grownup overnight in LB medium (supplemented with kanamycin 75 mg/l;spectinomycin 100 mg/l; chloramphenicol 25 mg/l) at 28° C. with shaking.The resulting culture (between 1% and 10% original volume) was theninoculated once more into LB (supplemented with kanamycin 75 mg/l;spectinomycin 100 mg/l; chloramphenicol 25 mg/l; 20 μM acetosyringone;and 10 mM MES) and grown overnight at 28° C. with shaking.

[0584] Plasmid P511, which contains the GUS reporter gene under thecontrol of the RD29a promoter, was transformed and amplified asdescribed for the 35S::CBF clone in this example. Plasmid vector pMEN 65was similarly innoculated and amplified to provide the vector control.

[0585] The overnight cultures were then pelleted by low-speedcentrifugation, 1500×g (3000 rpm in Sorvall RT7) for 25 minutes. Cellpellets were resuspended in ⅕ the original culture volume of freshlymade Infiltration Media (IM: 10 mM MES; 10 mM MgCl₂; 150 μMacetosyringone) and resuspended cell pellets were kept at ambienttemperature, with occasional mixing, for at least two hours before use.The resuspensed cell pellet was diluted with IM to attain an finalabsorption value at 600 nm of 1.0 in a 1 cm lightpath (A₆₀₀, O.D.=1.00).

[0586] Equal amounts of the P511 culture and the 35S::CBF culture weremixed by inversion to a final volume of 0.8 ml per tube.

[0587] Transient Transformation of Nicotiniana benthamiana Leaf Tissue

[0588] The plant was watered from the top two hours prior to theinfiltration procedure. Healthy symetrical leaves that had good turgorwere chosen. A total of ten ˜0.5 cm-diameter circles were marked on theaxial side of each leaf. A positive control was included on at least oneleaf of each plant. Negative controls were included in the assay onevery leaf. Control suspensions were made up for half the reporterconstruct mix and for the other half of the mix a binary vector withoutinsert was used.

[0589] The culture mix was taken up into a 1 ml disposable syringe andapproximately 0.1 ml was expeled onto each circle on the leaf. Theplants were then cultivated for five days until sample collection.

[0590] Extraction and GUS Assay

[0591] Each leaf disk was excised from the plant using a cork borer andplaced in 300 μl of Buffer 1 (50 mM Na+ Phosphate, pH 7.9; 16.7 mM EDTA)in a tube in a cluster tube rack (Catalogue No. 4413, Corning Inc.)which also contained a single stainless steel bead (Type 440C stainlessstell balls {fraction (3/16)}″, Catalogue No. 9529K13, McMaster-CarrSupply Co.).

[0592] One hundred microlitres of Buffer 2 (50 mM Na+ Phosphate, pH 7.9;50 mM β-mercaptoethanol) was then added to each tube, the tube wascapped, and the rack shaken in a model MM300 shaker-mixer (F. Kurt RetchGmbh & Co. K G, Haan, Germany) for 1.5 minutes at 28 beats/minute. Therack was rotated half a turn and shaken again for 1.5 minutes. The rackwas briefly centrifuged.

[0593] One hundred microlitres of Buffer 3 (50 mM Na+ Phosphate, pH 7.9;0.5% lauroylsarcosine, sodium salt; 0.5% TRITON X-100) was then added toeach tube and mixed by inversion. The tube was allowed to stand for twominutes at ambient temperature. The rack was centreifuged at 3000 rpm ina table top centrifuge for 15 minutes at 4° C.

[0594] Forty microliters of Extraction Buffer (3 parts Buffer 1 mixedwith 1 part Buffer 2 with 1 part Buffer 3) were added to each well in a96-well reaction plate (Microseal 96 Skirted V-bottom PolypropyleneMicroplate; Catalogue No. MSP-9601, Natural; MJ Research, Inc.) Tenmicrolitres of the supernatant from each tube was added to each well.

[0595] Ten miligrams of 4-methylumbelliferyl β-D-glucuronide (MUG;Catalogue No. M-5664, Sigma-Aldrich Chemical Co.) were disolved in 14.5ml of Extraction Buffer to approximately 2 mM. 50 μl of this MUGsolution was added to each sample in the reaction plate and the platewas incubated at 37° C. for one hour.

[0596] Two hundred microlitres of Stop Buffer (0.2 M Na₂CO₃) were addedto a black 96-well plate (Polyfiltronics 96-well, 300 μl, black,flat-bottom microplates; Catalogue No. PF030-PBX8, Phenix ResearchProducts, Hayward Calif.), then 8 μl of each reaction transferred toeach Stop Buffer plate well and mixed. The samples were then read usinga Synergy HT plate reader (BIOTECK Instruments, Inc., Winoosk, Vt.).

Example 22

[0597] Overexpression of CBF1 or CBF2 Increases Arabidopsis Drought orHigh Salt Tolerance

[0598] Soil studies were done by growing seedlings for 10 days withwater, and then letting the soil dry out (no further watering) until theplants were severely dehydrated. The soil was then watered and thenrecovery of the plants was measured. The transgenic lines werealternated with the control wild type lines, with a one-inch spacingbetween plants in two inches of soil. No detrimental effects wereobserved but the beneficial effects seen need further testing withdrought inducible promoter lines.

[0599] Two separate root elongation assays were performed to evaluatethe drought resistance phenotypes of the transgenic plants. First plantswere grown on MS agar plates for two weeks, and then transferred to MSagar plates containing either 300 mM mannitol or 150 mM NaCl. Thoseconcentrations were chosen because preliminary testing showed that wildtype plants showed the most dramatic reduction in root growth in thoseconditions. The root lengths were then measured after seven days, andthe data summarized in Table 14. The growth on sucrose (0.3% w/v), thenon-inhibition control and the growth on either salt or mannitol isshown. The control line lacking a CBF gene is the 643-3 line. When theratio of the CBF line to the 643-3 line (CBF/wild type) is significantlyabove 1.0, this is an indication of drought or salt tolerance.

[0600] From these, we concluded that the overexpression of the CBF genesdid provide growth benefit under high osmotic pressure and high salt,particularly for the CBF2 lines tested. TABLE 14 Mannitol (M) and Salt(S) Root Elongation Assay (mm) Ave Std CBF/wt CBF1#1 38.5 12.9 0.80643-3 48.2 10.4 CBF1#1 (S) 20.0 7.1 1.30 643-3 (S) 15.4 3.3 CBF1#1 (M)21.7 6.1 1.63 643-3 (M) 13.3 3.6 CBF1#6 43.7 16.8 0.94 643-3 46.5 14.7CBF1#6 (S) 16.5 4.0 1.03 643-3 (S) 16.0 3.8 CBF1#6 (M) 23.2 7.7 1.25643-3 (M) 18.5 3.6 CBF2#10 48.3 7.2 0.89 643-3 54.2 5.3 CBF2#10 (S) 15.83.7 1.08 643-3 (S) 14.7 2.6 CBF2#10 (M) 21.0 5.4 1.31 643-3 (M) 16.1 3.4CBF2#14 40.5 11.3 0.89 643-3 45.3 13.0 CBF2#14 (S) 21.5 3.9 1.50 643-3(S) 14.4 3.0 CBF2#14 (M) 24.0 5.3 1.67 643-3, (M) 14.4 2.6

Example 23

[0601] Identification of Transgenic Arabidopsis Plants that Express CBF3

[0602] Total soluble protein was obtained essentially as described(Gilmour et al. (1996) Plant Physiol. 111: 293-299) by grinding leafmaterial (about 100 mg) in 0.4 ml extraction buffer (50 mM PIPES pH 7.0,25 mM EDTA) containing 2.5% (w/v) polyvinyl-polypyrrolidone and removinginsoluble material by centrifugation (16000 g×20 min). Proteinconcentration in the supernatant was measured using the dye-bindingmethod of Bradford (1976) with BSA as the standard. Protein samples (50μg total protein) were fractionated by tricine SDS/PAGE (Schägger et al.(1987) Anal. Biochem. 166: 368-379) and transferred to 0.2 micronnitrocellulose membranes by electroblotting. COR15am and COR6.6 weredetected using the ECL kit (Amersham) with antiserum raised torecombinant COR15am and COR6.6 (Gilmour et al. (1996) Plant Physiol.111: 293-299).

[0603] Transgenic Arabidopsis plants that overexpress CBF3 at normalgrowth temperature were created by placing the CBF3 coding sequenceunder control of the cauliflower mosaic virus (CaMV) 35S promoter andtransforming the construct into Arabidopsis plants using the floral diptransformation procedure. Transgenic Arabidopsis plants that overexpressCBF3 were generated by transforming the chimeric genes into Arabidopsisecotype Ws-2 plants.

[0604] A 910 bp BamHI/HindIII fragment from a cDNA clone containing thewhole coding region of CBF3 (Gilmour et al. (1998) Plant J. 16: 433-442)was inserted into the BglII and HindIII sites of the binarytransformation vector pGA643. PGA643 has a CaMV 35S promoter and theterminator from gene 7 of pTiA6 (An (1988) “Binary Vectors”, in Gynheunget al., editors, (1995) Plant Molecular Biology Manual, Kluwer Acad.Publishers). The resulting plasmid, pMPS13, which contains the CBF3coding sequence under control of the CaMV 35S promoter, was transformedinto Agrobacterium tumefaciens strain GV3101 by electroporation (Konczet al. (1986) Mol. Gen. Gen. 204: 383). Arabidopsis plants weretransformed with plasmid pMPS13 or the transformation vector pGA643using the floral dip method (Clough et al. (1998) Plant J. 16: 735-743).Transformed plants were selected on the basis of kanamycin resistance.Homozygous T3 or T4 plants were used in all experiments.

[0605] Standard procedures were used for plasmid manipulations (Sambrooket al. (1989), supra). Prior to transformation, Arabidopsis thalianaseeds were sown at a density of ˜10 plants per 4″ pot onto Bactoplanting mix (Michigan Peat Co., Houston, Tex.) covered with fiberglassmesh (18 mm×18 mm). Plants were grown under continuous illumination(100-150 μE/m²/sec) at 20-22° C. with 65-70% relative humidity. Plantswere used when the primary inflorescences were approximately 10-12 cmhigh. The pots were then immersed upside down in the mixture ofAgrobacterium infiltration medium as described above (Clough et al.(1998) supra) for 2-3 seconds, and placed on their sides to allowdraining into a 1′×2′ flat surface covered with plastic wrap. After 24hours, the plastic wrap is removed and the pots were turned upright.Seeds were then collected from each transformation pot and analyzedfollowing the protocol described below

[0606] A construct comprising a cold-regulatable polypeptide gene codingsequence (CBF3) operably linked to a constitutive promoter wasgenerated.

[0607] Twenty-two independent lines were identified in which the T2plants segregated 3:1 for kanamycin resistance (the selectable markercarried on the transformation vector). These lines presumably carried asingle active T-DNA locus. The kanamycin resistant T2 plants were thenscreened by western analysis for constitutive expression of COR15a, atarget gene of the CBF transcriptional activators.

[0608] Three independent transgenic lines—A28, A30 and A40—wereidentified that produced the COR15am polypeptide at high levelsuniformly among plants grown at normal temperatures. Northern blotanalysis indicated that the transcript levels for CBF3 were about equalin non-cold acclimated and cold-treated A28, A30 and A40 plants and weremuch greater than those observed in either non-cold acclimated orcold-treated control plants (i.e. non-transformed plants or transgenicplants carrying the transformation vector without an insert). Thetranscript levels for two target COR genes, COR15a and COR6.6, were alsonearly equal in non-cold acclimated and cold-treated A28, A30 and A40plants and approximated the levels observed in cold-acclimated controlplants. Western blot analysis indicated that the proteins encoded byCOR15a and COR6.6 were present in both non-cold acclimated andcold-acclimated A28, A30 and A40 plants at 3 to 5 fold-higher levelsthan those found in cold-acclimated control plants.

Example 24

[0609] Overexpression of the CBF-Related Polypeptide in ArabidopsisAffects Physiological and Morphological Characteristics of the Plant

[0610] After the same number of days of vegetative growth at normaltemperature, physiological and morphological characteristics of theCBF3-expressing plants were compared with those of wild type plants. Thesize was less than that of the control plants. Additional differences ingrowth characteristics were also evident. One was that theCBF3-expressing plants had a pronounced prostrate growth habit; whereasthe leaves of the control plants generally had an upright stature, thoseof the transgenic plants laid flat to the soil. The CBF3-expressingplants also had much shorter petioles when compared to those of thecontrol plants and the leaves had a slight bluish-green tint. Also,there was a substantial difference in time to flowering between thecontrol and CBF3-expressing plants; i.e., control plants bolted andformed flowers well before the CBF3-expressing plants did. In oneexperiment, for instance, the control plants began to bolt at 17 dayswhile A40, A30 and A28 plants took 21, 26 and 28 days, respectively, toinitiate bolting (Table 15). TABLE 15 Effects of CBF3 Expression on Timeto Flowering and Rosette Leaf Number. Plants Time to Flowering (d)Rosette Leaves Per Plant Ws-2 17 4.5 B6 17 4.6 A40 21 6.0 A30 26 9.7 A2828 12.5

[0611] The CBF3-expressing plants went on to form flowers and set seed,though the final plant mass and seed yield was considerably less thanthat obtained with control plants. The lower yield of seed was due atleast in part to the CBF3-expressing plants producing fewer axillaryshoots. Significantly, the delay in flowering observed in theCBF3-expressing plants did not “simply” involve a slower overall growthrate, but appeared to involve a developmental delay in flowering. In oneexperiment, for instance, the control plants produced an average of 4.5and 4.6 leaves per rosette while the A40, A30, and A28 transgenic plantsproduced 6.0, 9.7, and 12.5 leaves per rosette, respectively (Table 15).

[0612] In some plants, including Arabidopsis, the transition toflowering is responsive to vernalization, a long period (weeks) of lowtemperature treatment. In “facultative” plants such as Arabidopsis, theeffect of vernalization is to shorten the time to flowering (themagnitude of the effect varies greatly among Arabidopsis ecotypes). IfCBF3 expression at warm temperature was fully mimicking exposure to lowtemperature, then one might think that if it had any effect onflowering, that it would decrease the transition time and cause acorresponding decrease in the number of rosette leaves per plant. Ourresults, however, indicate that CBF3 expression had the opposite effect;it increased the time to flowering and increased the number of rosetteleaves per plant.

[0613] When plants expressed a CBF-related polypeptide behind aninducible promoter, the plants were late flowering and their leaf numberwhen measured at the time of bolting (i.e. at a comparable developmentalstage) was increased compared to the control as was the above groundbiomass at the developmental stage as shown in FIGS. 27A through 27C.The Dreb2a::CBF1 plants grew more slowly, i.e. when plants of same agewere compared the biomass of the transgenics is slightly smaller (˜30%)than that of the control plants. But as the plants flowered later theygrow for a longer period of time prior to bolting and at thisdevelopmental stage they had more leaves and higher biomass than thecontrol plant at the same developmental stage (which the control plantreached a few days earlier).

Example 25

[0614] Vegetative Growth of Canola after Transformation with PlasmidsContaining CBF1, CBF2, or CBF3

[0615] Canola was transformed with a plasmid containing the ArabidopsisCBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987)Methods Enzymol. 253: 292). In these constructs the CBF genes wereexpressed constitutively under the CaMV 35S promoter. In addition, theCBF1 gene was cloned under the control of the Arabidopsis COR15 promoterin the same vector pGA643. Each construct was transformed intoAgrobacterium strain GV3101. Transformed agrobacteria was grown for 2days in Minimal AB medium containing the appropriate antibiotics.

[0616] Spring canola (B. napus cv. Westar) was transformed using theprotocol of Moloney (Moloney et. al. (1989) Plant Cell Reports 8: 238)with some modifications as described. Briefly, seeds were sterilized andplated on half strength MS medium, containing 1% sucrose. Plates wereincubated at 24° C. under 60-80 uE/m²s light using a16 hour light/8 hourdark photoperiod. Cotyledons from 4-5 day old seedlings were collected,the petioles cut and dipped into the Agrobacterium solution. The dippedcotyledons were placed on co-cultivation medium at a density of 20cotyledons/plate and incubated as described above for 3 days. Explantswere transferred to the same media, but containing 300 mg/l timentin(GlaxoSmithKline, PA) and thinned to 10 cotyledons/plate. After 7 daysexplants were transferred to Selection/Regeneration medium. Transferswere continued every 2-3 weeks (2 or 3 times) until shoots haddeveloped. Shoots were transferred to Shoot-Elongation medium every 2-3weeks. Healthy looking shoots were transferred to rooting medium. Oncegood roots had developed, the plants were placed into moist pottingsoil.

[0617] The transformed plants were then analyzed for the presence of theNPTII gene/kanamycin resistance by Elisa, using the Elisa NPTII kit from5Prime-3Prime Inc. (Boulder, Colo.). Approximately 70% of the screenedplants were NPTII positive. Only those plants were further analyzed.

[0618] After the same number of days of vegetative growth at normaltemperature, physiological and morphological characteristics of theCBF-expressing plants were compared with those of wild type plants. Ourresults indicated that CBF3 increased the time to flowering andincreased the number of rosette leaves per plant. This phenotype wasmost obvious in transgenic canola plants overexpressing CBF1, CBF2, orCBF3 under control of the 35S promoter.

Example 26

[0619] Overexpression of CBF1, CBF2, or CBF3 Affects Stress Tolerance inPlants

[0620] In addition to modification to the biomass of plants that occursdue to overexpression of CBF1, CBF2, or CBF3 genes shown in the aboveexamples, plants that have been transformed and overexpress these genesmay also be made more resistant to environmental stresses. Thus, whengrown under adverse conditions, the biomass of plant transformed withCBF1, CBF2, or CBF3 genes may be significantly greater than that of aplant that is more susceptible to the stress(es) and fails to thrive.

[0621] Overexpression of CBF1, CBF2, or CBF3 Increases FreezingTolerance in Arabidopsis.

[0622] Ws-2 and A30 seedlings were grown (13 days and 20 days,respectively) on Gamborg's B-5 medium containing 0.2% sucrose understerile conditions in Petri dishes. The plants were tested for freezingtolerance by first placing the plates at −2° C. in the dark for 24 hoursfollowed by ice nucleation with sterile ice chips for 3 hours. Thetemperature of the freezer was then turned down to −6° C. and the plateswere left at this temperature for an additional 24 hours. The plateswere taken from the freezer and placed at 4° C. in the dark for 18hours, followed by 2 days at 22° C. under cool white fluorescent lights(40-50 μmol m⁻² s⁻¹) with an 18 hour photoperiod. The plates were scored2 days later for freezing damage.

[0623] Electrolyte leakage freeze tests were performed essentially asdescribed (Uemura et al. (1995) Plant Physiol. 109: 15-30) with minormodifications. Tubes (16×125 mm) containing 3-4 leaves were placed in alow temperature bath set at −2° C. in a randomized design. Therandomization was performed with the aid of the SAS system (SASInstitute Inc, Cary N.C.). Ice chips were added to each tube after 1hour incubation. Each tube was capped with foam plugs and incubated afurther 1 hour at −2° C. The bath temperature was then lowered onedegree C every 20 minutes. Tubes were removed at each temperature andincubated an additional hour at that same temperature in a separatebath. Tubes were placed on ice after removal from the bath until alltubes had been removed. The samples were then thawed overnight at 2.5°C. and electrolyte leakage was measured as described (Gilmour et al.(1988) Plant Physiol. 87: 745-750).

[0624] Non-cold acclimated control plants were killed by freezing at −6°C. for 24 hours while non-cold acclimated CBF3-expressing plants werenot. Results for Ws-2 and A30 plants are shown in FIG. 26A. Electrolyteleakage tests indicated that the freezing tolerance of non-coldacclimated CBF3-expressing plants was about 3 to 4° C. greater than thatof the non-cold acclimated control plants. Specifically, non-coldacclimated control plants had an EL₅₀ (temperature that caused a 50%leakage of electrolytes) of about −4.5° C. while the three CBF3expressing lines had EL₅₀ values of about −8° C. (FIG. 26B).Significantly, the freezing tolerance of cold-acclimated CBF3-expressingplants was considerably greater than that of cold-acclimated controlplants. Control plants that had been cold-acclimated for 7 days had anEL₅₀ value of about −6° C. while 7 days cold-acclimated CBF3-expressingplants had EL₅₀ values of −11° C. or lower (FIGS. 26C and 26D).

[0625] Overexpression of CBF1, CBF2, or CBF3 Increases Salt Tolerance inCanola.

[0626] Canola was transformed with a plasmid containing the ArabidopsisCBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987)Methods Enzymol. 253: 292). In these constructs the CBF genes wereexpressed constitutively under the CaMV 35S promoter. In addition, theCBF1 gene was cloned under the control of the Arabidopsis COR15 promoterin the same vector pGA643. Each construct was transformed intoAgrobacterium strain GV3101. Transformed agrobacteria was grown for 2days in Minimal AB medium containing the appropriate antibiotics.

[0627] Spring canola (B. napus cv. Westar) was transformed using theprotocol of Moloney (Moloney et al. (1989) Plant Cell Reports 8: 238)with some modifications as described. Briefly, seeds were sterilized andplated on half strength MS medium, containing 1% sucrose. Plates wereincubated at 24° C. under 60-80 uE/m²s light using a16 hour light/8 hourdark photoperiod. Cotyledons from 4-5 day old seedlings were collected,the petioles cut and dipped into the Agrobacterium solution. The dippedcotyledons were placed on co-cultivation medium at a density of 20cotyledons/plate and incubated as described above for 3 days. Explantswere transferred to the same media, but containing 300 mg/L timentin(GlaxoSmithKline, PA) and thinned to 10 cotyledons/plate. After 7 daysexplants were transferred to Selection/Regeneration medium. Transferswere continued every 2-3 weeks (2 or 3 times) until shoots haddeveloped. Shoots were transferred to Shoot-Elongation medium every 2-3weeks. Healthy looking shoots were transferred to rooting medium. Oncegood roots had developed, the plants were placed into moist pottingsoil.

[0628] The transformed plants were then analyzed for the presence of theNPTII gene/kanamycin resistance by Elisa, using the Elisa NPTII kit from5Prime-3Prime Inc. (Boulder, Colo.). Approximately 70% of the screenedplants were NPTII positive. Only those plants were further analyzed.

[0629] From Northern blot analysis of the plants that were transformedwith the constitutively expressing constructs, showed expression of theCBF genes and all CBF genes were capable of inducing the Brassica napuscold-regulated gene BN115 (homolog of the Arabidopsis COR15 gene). Mostof the transgenic plants appear to exhibit a normal growth phenotype. Asexpected, the transgenic plants are more freezing tolerant than thewild-type plants. Using the electrolyte leakage of leaves test, thecontrol showed a 50% leakage at −2 to −3° C. Spring canola transformedwith either CBF1 or CBF2 showed a 50% leakage at −6 to −7° C. Springcanola transformed with CBF3 shows a 50% leakage at about −10 to −15° C.Winter canola transformed with CBF3 may show a 50% leakage at about −16to −20° C. Furthermore, if the spring or winter canola are coldacclimated the transformed plants may exhibit a further increase infreezing tolerance of at least −2° C.

[0630] To test salinity tolerance of the transformed plants, plants werewatered with 150 mM NaCl. Plants overexpressing CBF1, CBF2, or CBF3 grewbetter compared with plants that had not been transformed with CBF1,CBF2, or CBF3.

Example 27

[0631] Overexpression of CBF3 Affects Proline Metabolism in Arabidopsis

[0632] Proline levels in leaf samples were analyzed by methods describedin Example 5.

[0633] Under non-cold acclimating growth conditions, the free prolinelevels in the CBF3-expressing plants were about 5-fold higher than theywere in the control plants, levels which were about the same as thosefound in cold-acclimated control plants (FIG. 22). The proline levels inthe CBF3-expressing plants increased further (about 2-fold) upon coldacclimation and were 2-3 fold higher than those found in thecold-acclimated control plants (FIG. 22).

[0634] The proline biosynthetic enzyme Δ′-pyrroline-5-carboxylatesynthase has a key role in determining proline levels in plants (Yoshibaet al. (1997) Plant Cell Physiol. 38: 1095-1102). Because of this, andthat P5CS transcript levels have been shown to increase in Arabidopsisin response to low temperature (Xin et al. (1998) Proc. Natl. Acad. Sci.95: 7799-7804), it was of interest to determine whether P5CS transcriptlevels were elevated in the CBF3-expressing plants. Northern analysisindicated that they were; P5CS transcript levels were about 4 foldhigher in non-cold acclimated CBF3-expressing plants than they were innon-cold acclimated control plants and were about equal to those foundin the control plants that had been cold-treated for 1 day (FIG. 23).The P5CS transcript levels in 7-day cold-acclimated CBF3-expressingplants were 2 to 3 fold higher than in cold-acclimated control plants(FIG. 23), a finding that was consistent with the 2 to 3 fold higherlevels of proline found in the cold-acclimated CBF3-expressing plants(FIG. 22).

Example 28

[0635] Overexpression of CBF3 Affects Sugar Metabolism in Arabidopsis

[0636] Total soluble sugars (for example, sucrose, glucose, and fructoseamong others) were extracted from lyophilized leaf material as describedin Example 5. The results showed that CBF3 expression affected the sugarlevels in plants. Total soluble sugars in control and CBF3-expressingplants at both non-cold acclimating and cold acclimating temperatureswere measured. The results show (FIG. 24) that the levels of totalsugars in non-cold acclimated CBF3-expressing plants were about 3-foldgreater than those in non-cold acclimated control plants. Upon coldacclimation, sugar levels went up in both the control andCBF3-expressing plants about 2-fold, and remained about 3-fold higher inthe CBF3-expressing plants. Analysis of the sugars by HPLC indicatedthat CBF3 expression affected the levels of sucrose; in non-coldacclimated control plants, sucrose levels were about 0.3 μg/100 μg dryweight (DW), while in non-cold acclimated CBF3-expressing plants theywere about 1.5 μg/100 μg DW.

Example 29

[0637] Overexpression of CBF3 Affects Lipid Composition

[0638] Total lipids were extracted from Arabidopsis leaves and measuredas described in Example 5. The results indicated that CBF3 expressionaffected lipid composition. The representative results of Ws-2 and A28are presented in FIG. 25. They indicate that expression of CBF3 hadlittle or no effect on the relative amounts (mol %) of 16:0, 16:3, 18:2or 18:3 fatty acids in non-cold acclimated plants. Significantly, noappreciable change in the relative amounts of these fatty acids occurredduring cold acclimation either. Cold acclimation did result in sizabledecreases (30 to 50%) in the relative amounts of 16:1, 16:2, 18:0 and18:1 fatty acids and in three of these cases, specifically 16:1, 16:2,and 18:0 fatty acids, CBF3 expression caused similar decreases to occurin non-cold acclimated plants. In the case of 18:1 fatty acids, CBF3expression had an opposite effect from cold acclimation; it resulted ina slight increase in the relative levels of this fatty acid in non-coldacclimated transgenic plants and about a 50% increase in cold-acclimatedtransgenic plants. Taken together, these results indicate thatoverexpression of CBF3 has an effect on fatty acid composition and thatcertain of the changes mimic those that occur with cold acclimation.

Example 30

[0639] Overexpression of CBF3 Increases Arabidopsis Freezing Tolerance

[0640] Ws-2 and A30 seedlings were grown (13 days and 20 days,respectively) on Gamborg's B-5 medium containing 0.2% sucrose understerile conditions in Petri dishes. The plants were tested for freezingtolerance by first placing the plates at −2° C. in the dark for 24 hoursfollowed by ice nucleation with sterile ice chips for 3 hours. Thetemperature of the freezer was then turned down to −6° C. and the plateswere left at this temperature for an additional 24 hours. The plateswere taken from the freezer and placed at 4° C. in the dark for 18hours, followed by 2 days at 22° C. under cool white fluorescent lights(40-50 μmol m⁻² s⁻¹) with an 18 hour photoperiod. The plates were scored2 days later for freezing damage.

[0641] Electrolyte leakage freeze tests were performed essentially asdescribed (Uemura et al. (1995) Plant Physiol. 109: 15-30) with minormodifications. Tubes (16×125 mm) containing 3-4 leaves were placed in alow temperature bath set at −2° C. in a randomized design. Therandomization was performed with the aid of the SAS system (SASInstitute Inc, Cary N.C.). Ice chips were added to each tube after 1hour incubation. Each tube was capped with foam plugs and incubated afurther 1 hour at −2° C. The bath temperature was then lowered onedegree C. every 20 minutes. Tubes were removed at each temperature andincubated an additional hour at that same temperature in a separatebath. Tubes were placed on ice after removal from the bath until alltubes had been removed. The samples were then thawed overnight at 2.5°C. and electrolyte leakage was measured as described (Gilmour et al.(1988) Plant Physiol. 87: 745-750).

[0642] Non-cold acclimated control plants were killed by freezing at −6°C. for 24 hours while non-cold acclimated CBF3-expressing plants werenot. Results for Ws-2 and A30 plants are shown in FIG. 26A. Electrolyteleakage tests indicated that the freezing tolerance of non-coldacclimated CBF3-expressing plants was about 3 to 4° C. greater than thatof the non-cold acclimated control plants. Specifically, non-coldacclimated control plants had an EL₅₀ (temperature that caused a 50%leakage of electrolytes) of about −4.5° C. while the three CBF3expressing lines had EL₅₀ values of about −8° C. (FIG. 26B).Significantly, the freezing tolerance of cold-acclimated CBF3-expressingplants was considerably greater than that of cold-acclimated controlplants. Control plants that had been cold-acclimated for 7 days had anEL₅₀ value of about −6° C. while 7 days cold-acclimated CBF3-expressingplants had EL₅₀ values of −11° C. or lower (FIGS. 26C and 26D).

Example 31

[0643] Transformation and Expression in Arabidopsis Plants withCBF-Related Polynucleotides from other Plants

[0644] SEQ ID NOs: 294 through 318 were transformed into Arabidopsisplants as described in Examples 7 through 11. Transformed plants arescreened for traits as described in Examples 7 through 11.

Example 32

[0645] Transformation and Expression in Alfalfa Plants with CBFPolynucleotides

[0646] SEQ ID NOs: 294 through 318 were transformed into Medicago sativaplants as described. For alfalfa transformation a well establishedprotocol from Deborah Samac's laboratory was used which is based on asystem described by Austin et al., (Austin et al. (1995) Euphytica 85:381-393). This system gives a regeneration rate of callus piecesproducing embryos and later on transgenic plantlets of more than 75%.Transformation of the callus to the generation of transgenic plantletstook from between 9-12 weeks, and the transgenic plantlets were thenpropagated by cuttings. Leaf explants from the alfalfa cultivar Regen-SYare best suited for rapid production of embryos (Bingham (1991) CropSci. 31: 1098).

[0647] For transformation, young leaves (top second to fifth node) fromsoil grown plants were surface sterilized by a brief rinse in 70%ethanol followed by gentle agitation in a 20% bleach solution for 90seconds. After three rinses in sterile water, leaflet margins weretrimmed away and the remaining piece was cut in half. 50 leaf pieceswere generated and placed in 12 ml of liquid SH medium without hormones(Schenk and Hildebrandt (1972) Can. J. Bot. 50: 199-204). The day beforetransformation a 4 ml overnight culture (YEP liquid media containing 25mg/l rifampicin) of A. tumefaciens strain LBA4404 containing the binaryvector of interest was inoculated. For the transformation, 3 ml of thisovernight culture were added to the leaf pieces and incubated at roomtemperature for 10-15 minutes. After that, the leaf pieces were removed,blotted briefly on sterile filter paper, and placed on plates containingsolid callus-inducing B5 media with vitamins (Brown and Atanassov (1985)Plant Cell Tissue Organ Cult. 4:111-122): B5 salts and vitamins (Gamborget al. (1968) Exp. Cell Res. 50: 151-158), 30 g/l sucrose, 0.5 g/l KNO₃,0.25 g/l MgSO₄-7H₂O, 0.5 g/l proline, 798 mg/l L-glutamine, 99.6 mg/lserine, 0.48 mg/l adenine, 9.6 mg/l glutathione, 1 mg/l 2,4-D, 0.1 mg/lkinetin and 8 g/l Phytagar (Life Technologies, Inc.), pH 5.7). Theplates were sealed with gas-permeable tape (#394 3M Venting Tape) andincubated for 7 days at 24° C. with a light intensity of approximately100 μmol m⁻² sect⁻¹.

[0648] After 7 days co-cultivation the leaf pieces were rinsed 3-4 timesin sterile water, and transferred to fresh selection plates (B5 mediawith vitamins containing 25 mg/l kanamycin and 100 mg/l ticarcillin ortimentin). Calli and a few embryos formed after about two weeks, andwere then transferred to MMS medium (MMS: MS salts, 30 g/l sucrose,vitamins (Nitsch and Nitsch (1969) Science 163: 85-87), 100 mg/lmyo-inositol, 7 g/l phytagar plus 25 mg/l kanamycin and 100 mg/lticarcillin or timentin) and cultured for an additional 2-3 weeks. Ascotyledonary stage embryos grew out from the calli, they were cut offand transferred to fresh MMS medium with antibiotics for conversion toplantlets. Plantlets that formed a primary leaf were transferred toMagenta boxes containing fresh MMS media without kanamycin. This allowedfor further root and shoot development. After 2-5 weeks plants thatformed roots were removed from the boxes and placed in a test tubecontaining water. They were kept on the lab bench for 12-48 hours tocondition the plants and which reduced leaf loss due to desiccationafter transplanting. Individual plants were transplanted to a soil mixand maintained in a growth chamber under conditions to maximize leafproduction: 24° C./19° C. day/night temperature, 16/8 hours oflight/dark cycle with a light intensity of approximately 300 μmol m⁻²sec⁻¹). Plants were watered daily and were fertilized weekly with acomplete fertilizer.

[0649] Plants from individual lines were propagated by cuttings andtransferred to soil. Original transformants were maintained in Magentaboxes on selection, while the working plant material was generated bycuttings from the soil explants.

[0650] Up to 100 independent plants were tested by PCR for stableintegration of the transgene to ensure at least 20 independenttransgenic plants were generated per construct. As control forsuccessful setup of the transformation capabilities a 35s::intGUSconstruct was used. An empty vector control was included with the batchof 4 CBF constructs (total of 6 constructs). The initial transformants(at least 20 per construct) were propagated by cuttings after theydeveloped 5-8 internodes (roughly 7 weeks after transfer to soil). Eachcut internode was placed in vermiculite for 1-2 weeks for rootformation. 4-7 plants per transgenic line were transplanted in SC-10Super Cell Cone-tainers (Hummert; cones: 3.8 cm diameter, 21 cm depth;98 cones per 61×30.5 cm² tray) and grown under a 16/8 hours light/darkcycle either in the growth room at 24° C. or in a growth chamber at 24°C./19° C. day/night temperature (or constant at 4° C. for coldacclimation experiments) for further analysis.

[0651] Transformed plants are screened for traits as described inExamples 7 through 11.

[0652] While the present invention is disclosed by reference to thepreferred embodiments and examples detailed above, it is to beunderstood that these examples are intended in an illustrative ratherthan limiting sense, as it is contemplated that modifications willreadily occur to those skilled in the art, which modifications will bewithin the spirit of the invention and the scope of the appended claims.

1 332 1 905 DNA Arabidopsis thaliana CBF1 gene 1 aaaaagaatc tacctgaaaagaaaaaaaag agagagagat ataaatagct taccaagaca 60 gatatactat cttttattaatccaaaaaga ctgagaactc tagtaactac gtactactta 120 aaccttatcc agtttcttgaaacagagtac tctgatcaat gaactcattt tcagcttttt 180 ctgaaatgtt tggctccgattacgagcctc aaggcggaga ttattgtccg acgttggcca 240 cgagttgtcc gaagaaaccggcgggccgta agaagtttcg tgagactcgt cacccaattt 300 acagaggagt tcgtcaaagaaactccggta agtgggtttc tgaagtgaga gagccaaaca 360 agaaaaccag gatttggctcgggactttcc aaaccgctga gatggcagct cgtgctcacg 420 acgtcgctgc attagccctccgtggccgat cagcatgtct caacttcgct gactcggctt 480 ggcggctacg aatcccggagtcaacatgcg ccaaggatat ccaaaaagcg gctgctgaag 540 cggcgttggc ttttcaagatgagacgtgtg atacgacgac cacggatcat ggcctggaca 600 tggaggagac gatggtggaagctatttata caccggaaca gagcgaaggt gcgttttata 660 tggatgagga gacaatgtttgggatgccga ctttgttgga taatatggct gaaggcatgc 720 ttttaccgcc gccgtctgttcaatggaatc ataattatga cggcgaagga gatggtgacg 780 tgtcgctttg gagttactaatattcgatag tcgtttccat ttttgtacta tagtttgaaa 840 atattctagt tccttttttagaatggttcc ttcattttat tttattttat tgttgtagaa 900 acgag 905 2 213 PRTArabidopsis thaliana CBF1 polypeptide 2 Met Asn Ser Phe Ser Ala Phe SerGlu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Pro Gln Gly Gly Asp Tyr CysPro Thr Leu Ala Thr Ser Cys Pro Lys 20 25 30 Lys Pro Ala Gly Arg Lys LysPhe Arg Glu Thr Arg His Pro Ile Tyr 35 40 45 Arg Gly Val Arg Gln Arg AsnSer Gly Lys Trp Val Ser Glu Val Arg 50 55 60 Glu Pro Asn Lys Lys Thr ArgIle Trp Leu Gly Thr Phe Gln Thr Ala 65 70 75 80 Glu Met Ala Ala Arg AlaHis Asp Val Ala Ala Leu Ala Leu Arg Gly 85 90 95 Arg Ser Ala Cys Leu AsnPhe Ala Asp Ser Ala Trp Arg Leu Arg Ile 100 105 110 Pro Glu Ser Thr CysAla Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala 115 120 125 Ala Leu Ala PheGln Asp Glu Thr Cys Asp Thr Thr Thr Thr Asp His 130 135 140 Gly Leu AspMet Glu Glu Thr Met Val Glu Ala Ile Tyr Thr Pro Glu 145 150 155 160 GlnSer Glu Gly Ala Phe Tyr Met Asp Glu Glu Thr Met Phe Gly Met 165 170 175Pro Thr Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu Pro Pro Pro 180 185190 Ser Val Gln Trp Asn His Asn Tyr Asp Gly Glu Gly Asp Gly Asp Val 195200 205 Ser Leu Trp Ser Tyr 210 3 27 DNA Artificial Sequence MT50Description of Artificial Sequence C-repeat/DRE 3 gatcatttca tggccgacctgcttttt 27 4 28 DNA Artificial Sequence MT52 Description of ArtificialSequence C-repeat/DRE 4 cacaatttca agaattcact gctttttt 28 5 27 DNAArtificial Sequence MT80 Description of Artificial Sequence C-repeat/DRE5 gatcatttca tggtatgtct gcttttt 27 6 27 DNA Artificial Sequence MT125Description of Artificial Sequence C-repeat/DRE 6 gatcatttca tggaatcactgcttttt 27 7 27 DNA Artificial sequence MT68 Description of ArtificialSequence C-repeat/DRE 7 gatcacttga tggccgacct ctttttt 27 8 27 DNAArtificial Sequence MT66 Description of Artificial Sequence C-repeat/DRE8 gatcaatata ctaccgacat gagttct 27 9 25 DNA Artificial Sequence MT86Description of Artificial Sequence C-repeat/DRE 9 actaccgaca tgagttccaaaaagc 25 10 60 PRT Arabidopsis thaliana AP2 domain of 24 kDa polypeptide10 Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Ser Glu 1 510 15 Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 2025 30 Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 3540 45 Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 11 61 PRTNicotiana tabacum AP2 domain of tobacco DNA binding protein EREBP2 11His Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Phe Ala Ala Glu 1 5 1015 Ile Arg Asp Pro Ala Lys Asn Gly Ala Arg Val Trp Leu Gly Thr Tyr 20 2530 Glu Thr Ala Glu Glu Ala Ala Leu Ala Tyr Asp Lys Ala Ala Tyr Arg 35 4045 Met Arg Gly Ser Lys Ala Leu Leu Asn Phe Pro His Arg 50 55 60 12 651DNA Arabidopsis thaliana CBF2 gene 12 atgaactcat tttctgcctt ttctgaaatgtttggctccg attacgagtc tccggtttcc 60 tcaggcggtg attacagtcc gaagcttgccacgagctgcc ccaagaaacc agcgggaagg 120 aagaagtttc gtgagactcg tcacccaatttacagaggag ttcgtcaaag aaactccggt 180 aagtgggtgt gtgagttgag agagccaaacaagaaaacga ggatttggct cgggactttc 240 caaaccgctg agatggcagc tcgtgctcacgacgtcgccg ccatagctct ccgtggcaga 300 tctgcctgtc tcaatttcgc tgactcggcttggcggctac gaatcccgga atcaacctgt 360 gccaaggaaa tccaaaaggc ggcggctgaagccgcgttga attttcaaga tgagatgtgt 420 catatgacga cggatgctca tggtcttgacatggaggaga ccttggtgga ggctatttat 480 acgccggaac agagccaaga tgcgttttatatggatgaag aggcgatgtt ggggatgtct 540 agtttgttgg ataacatggc cgaagggatgcttttaccgt cgccgtcggt tcaatggaac 600 tataattttg atgtcgaggg agatgatgacgtgtccttat ggagctatta a 651 13 216 PRT Arabidopsis thaliana CBF2polypeptide 13 Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser AspTyr Glu 1 5 10 15 Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys LeuAla Thr Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg GluThr Arg His 35 40 45 Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly LysTrp Val Cys 50 55 60 Glu Leu Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp LeuGly Thr Phe 65 70 75 80 Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp ValAla Ala Ile Ala 85 90 95 Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala AspSer Ala Trp Arg 100 105 110 Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys GluIle Gln Lys Ala Ala 115 120 125 Ala Glu Ala Ala Leu Asn Phe Gln Asp GluMet Cys His Met Thr Thr 130 135 140 Asp Ala His Gly Leu Asp Met Glu GluThr Leu Val Glu Ala Ile Tyr 145 150 155 160 Thr Pro Glu Gln Ser Gln AspAla Phe Tyr Met Asp Glu Glu Ala Met 165 170 175 Leu Gly Met Ser Ser LeuLeu Asp Asn Met Ala Glu Gly Met Leu Leu 180 185 190 Pro Ser Pro Ser ValGln Trp Asn Tyr Asn Phe Asp Val Glu Gly Asp 195 200 205 Asp Asp Val SerLeu Trp Ser Tyr 210 215 14 651 DNA Arabidopsis thaliana CBF3 gene 14atgaactcat tttctgcttt ttctgaaatg tttggctccg attacgagtc ttcggtttcc 60tcaggcggtg attatattcc gacgcttgcg agcagctgcc ccaagaaacc ggcgggtcgt 120aagaagtttc gtgagactcg tcacccaata tacagaggag ttcgtcggag aaactccggt 180aagtgggttt gtgaggttag agaaccaaac aagaaaacaa ggatttggct cggaacattt 240caaaccgctg agatggcagc tcgagctcac gacgttgccg ctttagccct tcgtggccga 300tcagcctgtc tcaatttcgc tgactcggct tggagactcc gaatcccgga atcaacttgc 360gctaaggaca tccaaaaggc ggcggctgaa gctgcgttgg cgtttcagga tgagatgtgt 420gatgcgacga cggatcatgg cttcgacatg gaggagacgt tggtggaggc tatttacacg 480gcggaacaga gcgaaaatgc gttttatatg cacgatgagg cgatgtttga gatgccgagt 540ttgttggcta atatggcaga agggatgctt ttgccgcttc cgtccgtaca gtggaatcat 600aatcatgaag tcgacggcga tgatgacgac gtatcgttat ggagttatta a 651 15 216 PRTArabidopsis thaliana CBF3 polypeptide 15 Met Asn Ser Phe Ser Ala Phe SerGlu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Ser Ser Val Ser Ser Gly GlyAsp Tyr Ile Pro Thr Leu Ala Ser Ser 20 25 30 Cys Pro Lys Lys Pro Ala GlyArg Lys Lys Phe Arg Glu Thr Arg His 35 40 45 Pro Ile Tyr Arg Gly Val ArgArg Arg Asn Ser Gly Lys Trp Val Cys 50 55 60 Glu Val Arg Glu Pro Asn LysLys Thr Arg Ile Trp Leu Gly Thr Phe 65 70 75 80 Gln Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala Leu Ala 85 90 95 Leu Arg Gly Arg Ser AlaCys Leu Asn Phe Ala Asp Ser Ala Trp Arg 100 105 110 Leu Arg Ile Pro GluSer Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala 115 120 125 Ala Glu Ala AlaLeu Ala Phe Gln Asp Glu Met Cys Asp Ala Thr Thr 130 135 140 Asp His GlyPhe Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr 145 150 155 160 AlaGlu Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu Ala Met Phe 165 170 175Glu Met Pro Ser Leu Leu Ala Asn Met Ala Glu Gly Met Leu Leu Pro 180 185190 Leu Pro Ser Val Gln Trp Asn His Asn His Glu Val Asp Gly Asp Asp 195200 205 Asp Asp Val Ser Leu Trp Ser Tyr 210 215 16 18 DNA Nicotianatabacum MT117 primer 16 ttggcggcta cgaatccc 18 17 210 PRT Brassica napusCAN1 polypeptide 17 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser GlyLys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg IleTrp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His AspVal Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr AlaAsp Ser Ala Trp 50 55 60 Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys AspIle Gln Lys Ala 65 70 75 80 Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala GluLys Ser Asp Val Thr 85 90 95 Met Gln Asn Gly Gln Asn Met Glu Glu Thr ThrAla Val Ala Ser Gln 100 105 110 Ala Glu Val Asn Asp Thr Thr Thr Glu HisGly Met Asn Met Glu Glu 115 120 125 Ala Thr Ala Val Ala Ser Gln Ala GluVal Asn Asp Thr Thr Thr Asp 130 135 140 His Gly Val Asp Met Glu Glu ThrMet Val Glu Ala Val Phe Thr Gly 145 150 155 160 Glu Gln Ser Glu Gly PheAsn Met Ala Lys Glu Ser Thr Val Glu Ala 165 170 175 Ala Val Val Thr GluGlu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu 180 185 190 Trp Met Leu GluMet Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met 195 200 205 Leu Leu 21018 632 DNA Brassica napus CAN1 gene 18 cacccgatat accggggagt tcgtctgagaaagtcaggta agtgggtgtg tgaagtgagg 60 gaaccaaaca agaaatctag aatttggcttggaactttca aaacagctga gatggcagct 120 cgtgctcacg acgtcgctgc cctagccctccgtggaagag gcgcctgcct caattatgcg 180 gactcggctt ggcggctccg catcccggagacaacctgcc acaaggatat ccagaaggct 240 gctgctgaag ccgcattggc ttttgaggctgagaaaagtg atgtgacgat gcaaaatggc 300 cagaacatgg aggagacgac ggcggtggcttctcaggctg aagtgaatga cacgacgaca 360 gaacatggca tgaacatgga ggaggcaacggcagtggctt ctcaggctga ggtgaatgac 420 acgacgacgg atcatggcgt agacatggaggagacaatgg tggaggctgt ttttactggg 480 gaacaaagtg aagggtttaa catggcgaaggagtcgacgg tggaggctgc tgttgttacg 540 gaggaaccga gcaaaggatc ttacatggacgaggagtgga tgctcgagat gccgaccttg 600 ttggctgata tggcagaagg gatgctcctg cc632 19 36 DNA Artificial Sequence Dreb2a-reverse PCR primer 19gcccaagctt caagtttagt gagcactatg tgctcg 36 20 34 DNA Artificial SequenceDreb2a-forward PCR primer 20 ggaagatctc cttcccagaa acaacacaat ctac 34 2135 DNA Artificial Sequence P5CS-reverse PCR primer 21 gcccaagcttgtttcatttt ctccatgaag gagat 35 22 39 DNA Artificial SequenceP5CS-forward PCR primer 22 ggaagatctt atcgtcgtcg tcgtctacca aaaccacac 3923 32 DNA Artificial Sequence Rd22-reverse PCR primer 23 gctctaagcttcacaagggg ttcgtttggt gc 32 24 40 DNA Artificial Sequence Rd22-forwardPCR primer 24 ggggtacctt ttgggagttg gaatagaaat gggtttgatg 40 25 36 DNAArtificial Sequence Rd29a-reverse PCR primer 25 gcccaagctt aattttactcaaaatgtttt ggttgc 36 26 44 DNA Artificial Sequence Rd29a-forward PCRprimer 26 ccggtacctt tccaaagatt tttttctttc caatagaagt aatc 44 27 30 DNAArtificial Sequence Rd29b-reverse PCR primer 27 gcggaagctt cattttctgctacagaagtg 30 28 40 DNA Artificial Sequence Rd29b-forward PCR primer 28ccggtacctt tccaaagctg tgttttctct ttttcaagtg 40 29 42 DNA ArtificialSequence Rab18-reverse PCR primer 29 gcccaagctt caaattctga atattcacatatcaaaaaag tg 42 30 40 DNA Artificial Sequence Rab18-forward PCR primer30 ggaagatctg ttcttcttgt cttaagcaaa cactttgagc 40 31 41 DNA ArtificialSequence Cor47-reverse PCR primer 31 gcccaagctt tcgtctgtta tcatacaaggcacaaaacga c 41 32 42 DNA Artificial Sequence Cor47-forward PCR primer32 ggaagatcta gttaatcttg atttgattaa aagtttatat ag 42 33 25 DNAArtificial Sequence E9.1 primer PCR primer 33 caaactcagt aggattctggtgtgt 25 34 38 DNA Artificial Sequence cbf1-reverse 1 PCR primer 34ggaagatctt gaaacagagt actctgatca atgaactc 38 35 42 DNA ArtificialSequence cbf1-forward 1 PCR primer 35 cgcggatccc tcgtttctac aacaataaaataaaataaaa tg 42 36 37 DNA Artificial Sequence cbf1-reverse 2 PCR primer36 ggggtacctg aaacagagta ctctgatcaa tgaactc 37 37 41 DNA ArtificialSequence cbf1-forward 2 PCR primer 37 gctctagact cgtttctaca acaataaaataaaataaaat g 41 38 577 DNA Brassica juncea bjCBF1 gene 38 tttcaccctatctaccgggg agttcgcctg agaaagtcag gtaagtgggt gtgtgaagtg 60 agggagccaaacaagaaatc taggatttgg cttggaactt tcaaaaccgc agagatcgct 120 gctcgtgctcacgacgttgc cgccttagcc ctccgtggaa gagcggcctg tctcaacttc 180 gccgactcggcttggcggct ccgtatcccg gagacaactt gcgccaagga tatccagaag 240 gctgctgctgaagctgcgtt ggcttttggg gccgaaaaga gtgataccac gacgaatgat 300 caaggcatgaacatggagga gatgacggtg gtggcttctc aggctgaggt gagcgacacg 360 acgacatatcatggcctgga catggaggag actatggtgg aggctgtttt tgctgaggaa 420 cagagagaagggttttactt ggcggaggag acgacggtgg agggtgttgt tacggaggaa 480 cagagcaaagggttttatat gtacgaggag tggacgttcg ggatgcagtc ctttttggcc 540 gatatggctgaaggcatgct cttttcaaag ggcgaat 577 39 130 PRT Brassica juncea bjCBF1polypeptide 39 Leu Pro Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val CysGlu Val 1 5 10 15 Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly ThrPhe Lys Thr 20 25 30 Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala LeuAla Leu Arg 35 40 45 Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Ala TrpArg Leu Arg 50 55 60 Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys AlaAla Ala Glu 65 70 75 80 Ala Ala Leu Ala Phe Gly Ala Glu Lys Ser Asp ThrThr Thr Asn Asp 85 90 95 Gln Gly Met Asn Met Glu Glu Met Thr Ala Val AlaSer Gln Ala Glu 100 105 110 Val Ser Asp Thr Thr Thr Tyr His Gly Leu AspMet Glu Glu Thr Met 115 120 125 Val Asp 130 40 431 DNA Brassica junceabjCBF2 gene 40 catccgatct acaggggagt tcgtctgaga aaatcaggta agtgggtgtgtgaagtgagg 60 gaaccaaaca agagatctag gatctggctc ggtactttcc taaccgccgagatcgcagct 120 cgcgctcacg acgtcgccgc catagccctc cgtggcaaat ccgcatgtctcaatttcgct 180 gactcggctt ggcggctccg tatctcggag acaacatgcc ctaaggagattcagaaggct 240 gctgctgaag ccgcggtggc ttttcaggct gagctaaatg atacgacggccgatcatggc 300 cttgacgtgg aggagacgat cgtggaggct attttcacgg aggaaagcagcgaagggttt 360 tatatggacg aggagttcat gttcgggatg ccgaccttgt gggctagtatggcagaaggg 420 atgcttcttc c 431 41 143 PRT Brassica juncea bjCBF2polypeptide 41 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly LysTrp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Arg Ser Arg Ile TrpLeu Gly Thr 20 25 30 Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp ValAla Ala Ile 35 40 45 Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala AspSer Ala Trp 50 55 60 Arg Leu Arg Ile Ser Glu Thr Thr Cys Pro Lys Glu IleGln Lys Ala 65 70 75 80 Ala Ala Glu Ala Ala Val Ala Phe Gln Ala Glu LeuAsn Asp Thr Thr 85 90 95 Ala Asp His Gly Leu Asp Val Glu Glu Thr Ile ValGlu Ala Ile Phe 100 105 110 Thr Glu Glu Ser Ser Glu Gly Phe Tyr Met AspGlu Glu Phe Met Phe 115 120 125 Gly Met Pro Thr Leu Trp Ala Ser Met AlaGlu Gly Met Leu Leu 130 135 140 42 431 DNA Brassica juncea bjCBF3 gene42 catccaattt accgtggagt tcgtctgaga aaatcaggta agtgggtgtg tgaagtgagg 60gagccaaaca agaaatctag gatctggccc ggtactttcc taaccgccga gatcgcagct 120cgcgctcacg acgtcgccgc catagccctc cgtggcaaat ccgcatgtct caatttcgct 180gactcggctt ggcggctccg tatcccggag acaacatgcc ctaaggagat tcagaaggct 240gctgctgaag ccgcggtggc ttttcaggct gagctaaatg atacgacggc cgatcatggc 300cttgacgtgg aggagacgat cgtggaggct attttcacgg aggaaagcag cgaagggttt 360tatatggacg aggagttcat gttcgggatg ccgaccttgt gggctagtat ggcggagggc 420atgctccttc c 431 43 143 PRT Brassica juncea bjCBF3- polypeptide 43 HisPro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly Thr 20 25 30Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 35 40 45Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 50 55 60Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala 65 70 7580 Ala Ala Glu Ala Ala Val Ala Phe Gln Ala Glu Leu Asn Asp Thr Thr 85 9095 Ala Asp His Gly Leu Asp Val Glu Glu Thr Ile Val Glu Ala Ile Phe 100105 110 Thr Glu Glu Ser Ser Glu Gly Phe Tyr Met Ala Glu Glu Phe Met Phe115 120 125 Gly Met Pro Thr Leu Trp Ala Ser Val Ala Glu Gly Met Leu Leu130 135 140 44 425 DNA Brassica juncea bjCBF4 gene 44 catccaatctaccggggtgt tcgacagaga aactcaggga aatgggtttg tgaagttagg 60 gagcctaataagaaatctag gatctggtta gggacttttc cgaccgtcga aatggccgct 120 cgtgctcacgacgtcgccgc tttagccctt cgtggccgct ccgcttgtct taatttcgcc 180 gactcggcgtggtgtctacg gattcccgag tctacttgtc ctaaagagat tcagaaagct 240 gcggccgaagctgcaatggc gtttcagaac gagacggcta cgactgagac gactatggtt 300 gagggagtcataccggcgga ggagacggtg gggcagacgc gtgtggagac agcagaggag 360 aacggtgtgttttatatgga cgatccaagg tttcttgaga atatggcaga gggcatgttc 420 ctacc 425 45142 PRT Brassica juncea bjCBF4- polypeptide 45 His Pro Ile Tyr Arg GlyVal Arg Gln Arg Asn Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg GluPro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Pro Thr Val GluMet Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly ArgSer Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 50 55 60 Cys Leu Arg Ile ProGlu Ser Thr Cys Pro Lys Glu Ile Gln Lys Ala 65 70 75 80 Ala Ala Glu AlaAla Met Ala Phe Gln Asn Glu Glu Thr Ala Thr Thr 85 90 95 Glu Thr Thr MetVal Glu Gly Val Ile Pro Ala Glu Glu Thr Val Gly 100 105 110 Gln Thr ArgVal Glu Thr Ala Glu Glu Asn Gly Val Glu Tyr Met Asp 115 120 125 Asp ProArg Phe Leu Glu Asn Met Ala Glu Gly Met Leu Phe 130 135 140 46 632 DNABrassica napus bnCBF1 gene 46 cacccgatat accggggagt tcgtctgagaaagtcaggta agtgggtgtg tgaagtgagg 60 gaaccaaaca agaaatctag aatttggcttggaactttca aaacagctga gatggcagct 120 cgtgctcacg acgtcgctgc cctagccctccgtggaagag gcgcctgcct caattatgcg 180 gactcggctt ggcggctccg catcccggagacaacctgcc acaaggatat ccagaaggct 240 gctgctgaag ccgcattggc ttttgaggctgagaaaagtg atgtgacgat gcaaaatggc 300 cagaacatgg aggagacgac ggcggtggcttctcaggctg aagtgaatga cacgacgaca 360 gaacatggca tgaacatgga ggaggcaacggcagtggctt ctcaggctga ggtgaatgac 420 acgacgacgg atcatggcgt agacatggaggagacaatgg tggaggctgt ttttactggg 480 gaacaaagtg aagggtttaa catggcgaaggagtcgacgg tggaggctgc tgttgttacg 540 gaggaaccga gcaaaggatc ttacatggacgaggagtgga tgctcgagat gccgaccttg 600 ttggctgata tggcagaagg gatgctcctg cc632 47 210 PRT Brassica napus bnCBF1- polypeptide 47 His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr AlaGlu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg GlyArg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala Trp 50 55 60 Arg Leu Arg IlePro Glu Thr Thr Cys His Lys Asp Ile Gln Lys Ala 65 70 75 80 Ala Ala GluAla Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val Thr 85 90 95 Met Gln AsnGly Gln Asn Met Glu Glu Thr Thr Ala Val Ala Ser Gln 100 105 110 Ala GluVal Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 115 120 125 AlaThr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 130 135 140His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 145 150155 160 Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala165 170 175 Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp GluGlu 180 185 190 Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala GluGly Met 195 200 205 Leu Leu 210 48 876 DNA Brassica napus bnCBF2 gene 48accgctcgag caacaatgaa cacattccct gcttccactg aaatggttgg ctccgagaac 60gagtctccgg ttactacggt agtaggaggt gattattatc ccatgttggc ggcaagctgt 120ccgaagaagc cagcgggtag gaagaagttt caggagacac gtcaccccat ttaccgagga 180gttcgtctga gaaagtcagg taagtgggtg tgtgaagtga gggaaccaaa caagaaatct 240agaatttggc ccggaacttt caaaacagct gagatggcag ctcgtgctca cgacgtcgct 300gccctagccc tccgtggaag aggcgcctgc ctcaattatg cggactcggc ttggcggctc 360cgcatcccgg aaacaacctg ccacaaggat atccagaagg ctgctgctga agccgcattg 420gcttttgagg ctgagaaaag tgatgtgacg atgcaaaatg gcctgaacat ggaggagacg 480acggcggtgg cttctcaggc tgaagtgaat gacacgacga cagaacatgg catgaacatg 540gaggaggcaa cagcggtggc ttctcaggct gaggtgaatg acacgacgac agatcatggc 600gtagacatgg aggagacgat ggtggaggct gtttttacgg aggaacaaag tgaagggttc 660aacatggcgg aggagtcgac ggtggaggct gctgttgtta cggatgaact gagcaaagga 720ttttacatgg acgaggagtg gacgtacgag atgccgacct tgttggctga tatggcggca 780gggatgcttt tgccgccacc atctgtacaa tggggacata atgatgactt ggaaggagat 840gcggacatga acctctggag ttattaagga tccgcg 876 49 283 PRT Brassica napusbnCBF2- polypeptide 49 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val GlySer Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp TyrTyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg LysLys Phe Gln Glu Thr 35 40 45 Arg His Pro Ile Tyr Arg Gly Val Arg Leu ArgLys Ser Gly Lys Trp 50 55 60 Val Cys Glu Val Arg Glu Pro Asn Lys Lys SerArg Ile Trp Pro Gly 65 70 75 80 Thr Phe Lys Thr Ala Glu Met Ala Ala ArgAla His Asp Val Ala Ala 85 90 95 Leu Ala Leu Arg Gly Arg Gly Ala Cys LeuAsn Tyr Ala Asp Ser Ala 100 105 110 Trp Arg Leu Arg Ile Pro Glu Thr ThrCys His Lys Asp Ile Gln Lys 115 120 125 Ala Ala Ala Glu Ala Ala Leu AlaPhe Glu Ala Glu Lys Ser Asp Val 130 135 140 Thr Met Gln Asn Gly Leu AsnMet Glu Glu Thr Thr Ala Val Ala Ser 145 150 155 160 Gln Ala Glu Val AsnAsp Thr Thr Thr Glu His Gly Met Asn Met Glu 165 170 175 Glu Ala Thr AlaVal Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 180 185 190 Asp His GlyVal Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 195 200 205 Glu GluGln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu 210 215 220 AlaAla Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu 225 230 235240 Glu Trp Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 245250 255 Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Leu260 265 270 Glu Gly Asp Ala Asp Met Asn Leu Trp Ser Tyr 275 280 50 884DNA Brassica napus bnCBF3 gene 50 actacactca gccttatcca gtttttttcaaaagattttt caacaatgaa cacattccct 60 gcgtccactg aaatggttgg ctccgagaacgagtctccgg ttactacggt agcaggaggt 120 gattattatc ccatgttggc ggcaagctgtccgaagaagc cagcaggtag gaagaagttt 180 caggagacac gtcaccccat ttaccgaggagttcgtctga gaaagtcagg taagtgggtg 240 tgtgaagtga gggaaccaaa caagaaatctagaatttggc ccggaacttt caaaacagct 300 gagatggcag ctcgtgctca cgacgtcgctgccctagccc tccgtggaag aggcgcctgc 360 ctcaattatg cggactcggc ttggcggctccgcatcccgg agacaacctg ccacaaggat 420 atccagaagg ctgctgctga agccgcattggcttttgagg ctgagaaaag tgatgtgacg 480 atgcaaaatg gcctgaacat ggaggagacgacggcggtgg cttctcaggc tgaagtgaat 540 gacacgacga cagaacatgg catgaacatggaggaggcaa cggcagtggc ttctcaggct 600 gaggtgaatg acacgacgac ggatcatggcgtagacatgg aggagacaat ggtggaggct 660 gtttttactg gggaacaaag tgaagggtttaacatggcga aggagtcgac ggtggaggct 720 gctgttgtta cggaggaacc gagcaaaggatcttacatgg acgaggagtg gatgctcgag 780 atgccgacct tgttggctga tatggcggaagggatgcttt tgccgccgcc gtccgtacaa 840 tggggacaga atgatgactt cgaaggagatgctgacatga acct 884 51 279 PRT Brassica napus bnCBF3- polypeptide 51 MetAsn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 1 5 10 15Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 50 55 60Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly 65 70 7580 Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 85 9095 Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 100105 110 Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys115 120 125 Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser AspVal 130 135 140 Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala ValAla Ser 145 150 155 160 Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His GlyMet Asn Met Glu 165 170 175 Glu Ala Thr Ala Val Ala Ser Gln Ala Glu ValAsn Asp Thr Thr Thr 180 185 190 Asp His Gly Val Asp Met Glu Glu Thr MetVal Glu Ala Val Phe Thr 195 200 205 Gly Glu Gln Ser Glu Gly Phe Asn MetAla Lys Glu Ser Thr Val Glu 210 215 220 Ala Ala Val Val Thr Glu Glu ProSer Lys Gly Ser Tyr Met Asp Glu 225 230 235 240 Glu Trp Met Leu Glu MetPro Thr Leu Leu Ala Asp Met Ala Glu Gly 245 250 255 Met Leu Leu Pro ProPro Ser Val Gln Trp Gly Gln Asn Asp Asp Phe 260 265 270 Glu Gly Asp AlaAsp Met Asn 275 52 874 DNA Brassica napus bnCBF4 gene 52 gtaattcgattaccgctcga gtacttacta tactacactc agccttatcc agtttttcaa 60 aagaagttttcaactatgaa ctcagtctct actttttctg aacttcttgg ctctgagaac 120 gagtctccggtaggtggtga ttactgtccc atgttggcgg cgagctgtcc gaagaagccg 180 gcgggtaggaagaagtttcg ggagacacgt caccccattt accgaggagt tcgccttaga 240 aaatcaggtaagtgggtgtg tgaagtgagg gaaccaaaca aaaaatctag gatttggctc 300 ggaactttcaaaacagctga gatcgcagct cgtgctcacg acgtcgccgc cttagctctc 360 cgtggaagaggcgcctgcct caacttcgcc gactcggctt ggcggctccg tatcccggag 420 acaacctgcgccaaggatat ccagaaggct gctgctgaag ccgcattggc ttttgaggcc 480 gagaagagtgataccacgac gaatgatcat ggcatgaaca tggcttctca ggccgaggtt 540 aatgacacaacggatcatgg cctggacatg gaggagacga tggtggaggc tgtttttact 600 gaggagcagagagacgggtt ttacatggcg gaggagacga cggtggaggg tgttgttccg 660 gaggaacagatgagcaaagg gttttacatg gacgaggagt ggatgttcgg gatgccgacc 720 ttgttggctgatatggcggc agggatgctc ttaccgccgc cgtccgtaca atggggacat 780 aatgatgacttcgaaggaga tgttgacatg aacctctgga attattagta ctcatatttt 840 tttaaattattttttgaacg aataatattt tatt 874 53 250 PRT Brassica napus bnCBF4-polypeptide 53 Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser GluAsn Glu 1 5 10 15 Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala AlaSer Cys Pro 20 25 30 Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr ArgHis Pro Ile 35 40 45 Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp ValCys Glu Val 50 55 60 Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly ThrPhe Lys Thr 65 70 75 80 Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala AlaLeu Ala Leu Arg 85 90 95 Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser AlaTrp Arg Leu Arg 100 105 110 Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile GlnLys Ala Ala Ala Glu 115 120 125 Ala Ala Leu Ala Phe Glu Ala Glu Lys SerAsp Thr Thr Thr Asn Asp 130 135 140 His Gly Met Asn Met Ala Ser Gln AlaGlu Val Asn Asp Thr Thr Asp 145 150 155 160 His Gly Leu Asp Met Glu GluThr Met Val Glu Ala Val Phe Thr Glu 165 170 175 Glu Gln Arg Asp Gly PheTyr Met Ala Glu Glu Thr Thr Val Glu Gly 180 185 190 Val Val Pro Glu GluGln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu 195 200 205 Trp Met Phe GlyMet Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met 210 215 220 Leu Leu ProPro Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe Glu 225 230 235 240 GlyAsp Val Asp Met Asn Leu Trp Asn Tyr 245 250 54 898 DNA Brassica napusbnCBF5 gene 54 aataaatatc ttatcaaacc agtcagaaca gagatcttgt tacttactatactacactca 60 gccttatcca gttttcaaaa aaagtattca acgatgaact cagtctctactttttctgaa 120 ctgctccgct ccgagaacga gtctccggtt aatacggaag gtggtgattacattttggcg 180 gcgagctgtc ccaagaaacc tgctggtagg aagaagtttc aggagacacgccaccccatt 240 tacagaggag ttcgtctgag gaagtcaggt aagtgggtgt gtgaagtgagggaaccaaac 300 aagaaatcta gaatttggct cggaactttc aaaacagctg agatcgcagctcgtgctcac 360 gacgttgccg ccttagctct ccgtggaaga ggcgcctgcc tcaacttcgccgactcggct 420 tggcggctcc gtatcccgga gacgacctgc gccaaggata tccagaaggctgctgctgaa 480 gccgcattgg cttttgaggc cgagaagagt gataccacga cgaatgatcatggcatgaac 540 atggcttctc aggttgaggt taatgacacg acggatcatg acctggacatggaggagacg 600 atagtggagg ctgtttttag ggaggaacag agagaagggt tttacatggcggaggagacg 660 acggttgtgg gtgttgttcc ggaggaacag atgagcaaag ggttttacatggacgaggag 720 tggatgttcg ggatgccgac cttgttggct gatatggcgg cagggatgctcttaccgctg 780 ccgtccgtac aatggggaca taatgatgac ttcgaaggag atgctgacatgaacctctgg 840 aattattagt actcatattt ttttaaatta ttttttgaac gaataatattttattgaa 898 55 251 PRT Brassica napus bnCBF5- polypeptide 55 Met AsnSer Val Ser Thr Phe Ser Glu Leu Leu Arg Ser Glu Asn Glu 1 5 10 15 SerPro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 20 25 30 ProLys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg His Pro 35 40 45 IleTyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu 50 55 60 ValArg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys 65 70 75 80Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 85 90 95Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 100 105110 Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 115120 125 Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn130 135 140 Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn Asp ThrThr 145 150 155 160 Asp His Asp Leu Asp Met Glu Glu Thr Ile Val Glu AlaVal Phe Arg 165 170 175 Glu Glu Gln Arg Glu Gly Phe Tyr Met Ala Glu GluThr Thr Val Val 180 185 190 Gly Val Val Pro Glu Glu Gln Met Ser Lys GlyPhe Tyr Met Asp Glu 195 200 205 Glu Trp Met Phe Gly Met Pro Thr Leu LeuAla Asp Met Ala Ala Gly 210 215 220 Met Leu Leu Pro Leu Pro Ser Val GlnTrp Gly His Asn Asp Asp Phe 225 230 235 240 Glu Gly Asp Ala Asp Met AsnLeu Trp Asn Tyr 245 250 56 1132 DNA Brassica napus bnCBF6 gene 56gattaccgct cgagtactta ctatactaca ctcagcctta tccagttttt ctcaaaagat 60ttttcaacaa tgaacacatt ccctgcttcc actgaaatgg ttggctccga gaacgagtct 120ccggttacta cggtagtagg aggtgattat tatcccatgt tggcggcaag ctgtccgaag 180aagccagcgg gtaggaagaa gtttcaggag acacgtcacc ccatttaccg aggagttcgt 240ctgagaaagt caggtaagtg ggtgtgtgaa gtgagggaac caaacaagaa atctagaatt 300tggcttggaa ctttcaaaac agctgagatg gcagctcgtg ctcacgacgt ggctgcccta 360gccctccgtg gaagaggcgc ctgcctcaat tatgcggact cggcttcgcg gctccgcatc 420ccggagacaa cctgccacaa ggatatccag aaggctgctg ctgaagccgc attggctttt 480gaggctgaga aaagtgatgt gacgatggag gagacgatgg cggtggcttc tcaggctgaa 540gtgaatgaca cgacgacaga tcatggcatg aacatggagg aggcaacagc ggtggcttct 600caggctgagg tgaatgacac gacgacagat catggcgtag acatggagga gacgatggtg 660gaggctgttt ttacggagga acaaagtgaa gggttcaaca tggcggagga gtcgacggtg 720gaggctgctg ttgttacgga tgaactgagc aaaggatttt acatggacga ggagtggacg 780tacgagatgc cgaccttgtt ggctgatatg gcggcaggga tgcttttgcc gccaccatct 840gtacaatggg gacataatga tgacttggaa ggagatgctg acatgaacct ctggaattat 900taatactcgt gttttaaaaa ttatacattg tgcaataata ttttatcgaa tttctaattc 960tgcctttaac ttttaatggg gatctttatt agtgtaggaa acgagtgtaa atgttccgcc 1020gtggtgttgt caaaatgctg attatttttt gtgtgcagca taatcacgtt tggtttcctt 1080tacactccaa atttagttga aatacaaata gaatagaaaa gtgaaaaaat gt 1132 57 277PRT Brassica napus bnCBF6- polypeptide 57 Met Asn Thr Phe Pro Ala SerThr Glu Met Val Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr ValVal Gly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys LysPro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp 50 55 60 Val Cys Glu Val Arg GluPro Asn Lys Lys Ser Arg Ile Trp Leu Gly 65 70 75 80 Thr Phe Lys Thr AlaGlu Met Ala Ala Arg Ala His Asp Val Ala Ala 85 90 95 Leu Ala Leu Arg GlyArg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 100 105 110 Ser Arg Leu ArgIle Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 115 120 125 Ala Ala AlaGlu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 130 135 140 Thr MetGlu Glu Thr Met Ala Val Ala Ser Gln Ala Glu Val Asn Asp 145 150 155 160Thr Thr Thr Asp His Gly Met Asn Met Glu Glu Ala Thr Ala Val Ala 165 170175 Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met 180185 190 Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu Glu Gln Ser Glu Gly195 200 205 Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val Val ThrAsp 210 215 220 Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr TyrGlu Met 225 230 235 240 Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met LeuLeu Pro Pro Pro 245 250 255 Ser Val Gln Trp Gly His Asn Asp Asp Leu GluGly Asp Ala Asp Met 260 265 270 Asn Leu Trp Asn Tyr 275 58 768 DNABrassica napus bnCBF7 gene 58 agtgatgttt ttcaaaagaa gttttcaactatgaactcag tctctacttt ttctgaactt 60 cttggctctg agaacgagtc tccggtaggtggtgattact gtcccatgtt ggcggcgagc 120 tgtccgaaga agccggcggg taggaagaagtttcgggaga cacgtcaccc catttaccga 180 ggagttcgcc ttagaaaatc aggtaagtgggtgtgtgaag tgagggagcc aaacaagaaa 240 tctaggattt ggctcggtac tttcctaacagccgagatcg cagcccgtgc tcacgacgtc 300 gccgccatag ccctccgcgg caaatcagcttgtctcaatt ttgccgactc cgcttggcgg 360 ctccgtatcc cggagacaac atgccccaaggagattcaga aggcggctgc tgaagccgcg 420 gtggctttta aggctgagat aaataatacgacggcggatc atggcattga cgtggaggag 480 acgatcgttg aggctatttt cacggaggaaaacaacgatg gtttttatat ggacgaggag 540 gagtccatgt tcgggatgcc ggccttgttggctagtatgg ctgaaggaat gcttttgccg 600 cctccgtccg tacaattcgg acatacctatgactttgacg gagatgctga cgtgtccctt 660 tggagttatt agtacaaaga ttttttatttccatttttgg tataatactt ctttttgatt 720 ttcggattct acctttttat gggtatcattttttttttag gaaacggg 768 59 213 PRT Brassica napus bnCBF7 polypeptide 59Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 1 5 1015 Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 20 2530 Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 35 4045 Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 50 5560 Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Leu Thr 65 7075 80 Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg 8590 95 Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg100 105 110 Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala Ala AlaGlu 115 120 125 Ala Ala Val Ala Phe Lys Ala Glu Ile Asn Asn Thr Thr AlaAsp His 130 135 140 Gly Ile Asp Val Glu Glu Thr Ile Val Glu Ala Ile PheThr Glu Glu 145 150 155 160 Asn Asn Asp Gly Phe Tyr Met Asp Glu Glu GluSer Met Phe Gly Met 165 170 175 Pro Ala Leu Leu Ala Ser Met Ala Glu GlyMet Leu Leu Pro Pro Pro 180 185 190 Ser Val Gln Phe Gly His Thr Tyr AspPhe Asp Gly Asp Ala Asp Val 195 200 205 Ser Leu Trp Ser Tyr 210 60 953DNA Brassica napus bnCBF8 gene 60 accgctcgag caacaatgaa cacattccctgcttccactg aaatggttgg ctccgagaac 60 gagtctccgg ttactacggt agcaggaggtgattattatc ccatgttggc ggcaagctgt 120 ccgaagaagc cagcgggtag gaagaagtttcaggagacac gtcaccccat ttaccgagga 180 gttcgtctga gaaagtcagg taagtgggtgtgtgaagtga gggaaccaaa caagaaatct 240 agaatttggc ttggaacttt caaaacagctgagatggcag ctcgtgctca cgacgtggct 300 gccctagccc tccgtggaag aggcgcctgcctcaattatg cggactcggc ttcgcggctc 360 cgcatcccgg agacaacctg ccacaaggatatccagaagg ctgctgctga agccgcattg 420 gcttttgagg ctgagaaaag tgatgtgacgatggaggaga cgatggcggt ggcttctcag 480 gctgaagtga atgacacgac gacagatcatggcatgaaca tggaggaggc aacggcagtg 540 gcttctcagg ctgaggtgaa tgacacgacgacggatcatg gcgtagacat ggaggagaca 600 atggtggagg ctgtttttac tggggaacaaagtgaagggt ttaacatggc gaaggagtcg 660 acggtggagg ctgctgttgt tacggaggaaccgagcaaag gatcttacat ggacgaggag 720 tggatgctcg agatgccgac cttgttggctgatatggcgg aagggatgct tttgccgccg 780 ccgtccgtac aatggggaca gaatgatgacttcgaaggag atgcggacat gaacctctgg 840 agttattaat actcgtattt ttaaaattatttattgtgca ataatttttt atcgaatttc 900 gaattctgcc tttaattttt aatggggatctttatttgcc aaaaaaaaaa aaa 953 61 277 PRT Brassica napus bnCBF8polypeptide 61 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser GluAsn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr ProMet Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys PheGln Glu Thr 35 40 45 Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp 50 55 60 Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg IleTrp Leu Gly 65 70 75 80 Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala 85 90 95 Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn TyrAla Asp Ser Ala 100 105 110 Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys HisLys Asp Ile Gln Lys 115 120 125 Ala Ala Ala Glu Ala Ala Leu Ala Phe GluAla Glu Lys Ser Asp Val 130 135 140 Thr Met Glu Glu Thr Met Ala Val AlaSer Gln Ala Glu Val Asn Asp 145 150 155 160 Thr Thr Thr Asp His Gly MetAsn Met Glu Glu Ala Thr Ala Val Ala 165 170 175 Ser Gln Ala Glu Val AsnAsp Thr Thr Thr Asp His Gly Val Asp Met 180 185 190 Glu Glu Thr Met ValGlu Ala Val Phe Thr Gly Glu Gln Ser Glu Gly 195 200 205 Phe Asn Met AlaLys Glu Ser Thr Val Glu Ala Ala Val Val Thr Glu 210 215 220 Glu Pro SerLys Gly Ser Tyr Met Asp Glu Glu Trp Met Leu Glu Met 225 230 235 240 ProThr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu Pro Pro Pro 245 250 255Ser Val Gln Trp Gly Gln Asn Asp Asp Phe Glu Gly Asp Ala Asp Met 260 265270 Asn Leu Trp Ser Tyr 275 62 889 DNA Brassica napus bnCBF9 gene 62ctagtgatta ccgctcgagc aacaatgaac acattccctg cttccactga aatggttggc 60tccgagaacg agtctccggt tactacggta gcaggaggtg attattatcc catgttggcg 120gcaagctgtc cgaagaagcc agcgggtagg aagaagtttc aggagacacg tcaccccatt 180taccgaggag ttcgtctgag aaagtcaggt aagtgggtgt gtgaagtgag ggaaccaaac 240aagaaatcta gaatttggcc cggaactttc aaaacagctg agatggcagc tcgtgctcac 300gacgtcgctg ccctagccct ccgtggaaga ggcgcccgcc tcaattatgc ggactcagct 360tggcggctcc gcatcccgga gacaacctgc cacaaggata tccagaaggc tgctgctgaa 420gccgcattgg cttttgaggc tgagaaaagt gatgtgacga tgcaaaatgg cctgaacatg 480gaggagacga cggcggtggc ttctcaggct gaagtgaatg acacgacgac agaacatggc 540atgaacatgg aggaggcaac ggcagtggct tctcaggctg aggtgaatga cacgacgacg 600gatcatggcg tagacatgga ggagacaatg gtggaggctg tttttactgg ggaacaaagt 660gaagggttta acatggcgaa ggagtcgacg gtggaggctg ctgttgttac ggaggaaccg 720agcaaaggat cttacatgga cgaggagtgg atgctcgaga tgccgacctt gttggctgat 780atggcggaag ggatgctttt gccgccgccg tccgtacaat ggggacagaa tgatgacttc 840gaaggagatg cgcacatgaa cctctggagt tattaaggat ccgcgaatc 889 63 283 PRTBrassica napus bnCBF9 polypeptide 63 Met Asn Thr Phe Pro Ala Ser Thr GluMet Val Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Ala GlyGly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro AlaGly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg His Pro Ile Tyr Arg Gly ValArg Leu Arg Lys Ser Gly Lys Trp 50 55 60 Val Cys Glu Val Arg Glu Pro AsnLys Lys Ser Arg Ile Trp Pro Gly 65 70 75 80 Thr Phe Lys Thr Ala Glu MetAla Ala Arg Ala His Asp Val Ala Ala 85 90 95 Leu Ala Leu Arg Gly Arg GlyAla Arg Leu Asn Tyr Ala Asp Ser Ala 100 105 110 Trp Arg Leu Arg Ile ProGlu Thr Thr Cys His Lys Asp Ile Gln Lys 115 120 125 Ala Ala Ala Glu AlaAla Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 130 135 140 Thr Met Gln AsnGly Leu Asn Met Glu Glu Thr Thr Ala Val Ala Ser 145 150 155 160 Gln AlaGlu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 165 170 175 GluAla Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 180 185 190Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 195 200205 Gly Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu 210215 220 Ala Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu225 230 235 240 Glu Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met AlaGlu Gly 245 250 255 Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly Gln AsnAsp Asp Phe 260 265 270 Glu Gly Asp Ala His Met Asn Leu Trp Ser Tyr 275280 64 563 DNA Brassica oleracea boCBF1 gene 64 caccctatct accggggagttcgcctgaga aagtcaggta agtgggtgtg tgaagtgagg 60 gagccaaaca agaaatctaggatttggctt ggaactttca aaaccgcaga gatcgctgct 120 cgtgctcacg acgttgccgccttagccctc cgtggaagag cggcctgtct caacttcgcc 180 gactcggctt ggcggctccgtatcccggag acaacttgcg ccaaggatat ccagaaggct 240 gctgctgaag ctgcgttggcttttggggcc gaaaagagtg ataccacgac gaatgatcaa 300 ggcatgaaca tggaggagatgacggtggtg gcttctcagg ctgaggtgag cgacacgacg 360 acatatcatg gcctggacatggaggagact atggtggagg ctgtttttgc tgaggaacag 420 agagaagggt tttacttggcggaggagacg acggtggagg gtgttgttac ggaggaacag 480 agcaaagggt tttatatggacgaggagtgg acgttcggga tgcagtcctt tttggccgat 540 atggctgaag gcatgctctttcc 563 65 188 PRT Brassica oleracea boCBF1 polypeptide 65 His Pro IleTyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys GluVal Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe LysThr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala LeuArg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 50 55 60 Arg LeuArg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala 65 70 75 80 AlaAla Glu Ala Ala Leu Ala Phe Gly Ala Glu Lys Ser Asp Thr Thr 85 90 95 ThrAsn Asp Gln Gly Met Asn Met Glu Glu Met Thr Val Val Ala Ser 100 105 110Gln Ala Glu Val Ser Asp Thr Thr Thr Tyr His Gly Leu Asp Met Glu 115 120125 Glu Thr Met Val Glu Ala Val Phe Ala Glu Glu Gln Arg Glu Gly Phe 130135 140 Tyr Leu Ala Glu Glu Thr Thr Val Glu Gly Val Val Thr Glu Glu Gln145 150 155 160 Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Phe Gly MetGln Ser 165 170 175 Phe Leu Ala Asp Met Ala Glu Gly Met Leu Phe Pro 180185 66 533 DNA Brassica oleracea boCBF2 gene 66 gaaacataga tctttgtacttactatactt caccttatcc agttttattt ttttatttat 60 aaagagtttt caacaatgacctcattttct accttttctg aactgttggg ctccgagcat 120 gagtctccgg ttacattaggcgaagagtat tgtccgaagc tggccgcaag ctgtccgaag 180 aaaccagccg gccggaagaagtttcgagag acgcgtcacc cagtttacag aggagttcgt 240 ctgagaaact caggtaagtgggtgtgtgaa gtgagggagc caaacaagaa atctaggatt 300 tggctcggta ctttcctaacagccgagatc gcagcccgtg ctcacgacgt cgccgccata 360 gccctccgcg gcaaatcagcttgtctcaat tttgccgact ccgcttggcg gctccgtatc 420 ccggagacaa catgccccaaggagattcag aaggcggctg ctgaagccgc ggtggctttt 480 aaggctgaga taaataatacgacggcggat cacggcctcg acatggaaga gac 533 67 152 PRT Brassica oleraceaboCBF2 polypeptide 67 Met Thr Ser Phe Ser Thr Phe Ser Glu Leu Leu GlySer Glu His Glu 1 5 10 15 Ser Pro Val Thr Leu Gly Glu Glu Tyr Cys ProLys Leu Ala Ala Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys PheArg Glu Thr Arg His 35 40 45 Pro Val Tyr Arg Gly Val Arg Leu Arg Asn SerGly Lys Trp Val Cys 50 55 60 Glu Val Arg Glu Pro Asn Lys Lys Ser Arg IleTrp Leu Gly Thr Phe 65 70 75 80 Leu Thr Ala Glu Ile Ala Ala Arg Ala HisAsp Val Ala Ala Ile Ala 85 90 95 Leu Arg Gly Lys Ser Ala Cys Leu Asn PheAla Asp Ser Ala Trp Arg 100 105 110 Leu Arg Ile Pro Glu Thr Thr Cys ProLys Glu Ile Gln Lys Ala Ala 115 120 125 Ala Glu Ala Ala Val Ala Phe LysAla Glu Ile Asn Asn Thr Thr Ala 130 135 140 Asp His Gly Leu Asp Met GluGlu 145 150 68 887 DNA Brassica oleracea boCBF3 gene 68 actcagccttatccagtttt tctcaaaaga tttttcaaca atgaacacat tccctgcttc 60 cactgaaatggttggctccg agaacgagtc tccggttact acggtagtag gaggtgatta 120 ttatcccatgttggcggcaa gctgtccgaa gaagccagcg ggtaggaaga agtttcagga 180 gacacgtcaccccatttacc gaggagttcg tctgagaaag tcaggtaagt gggtgtgtga 240 agtgagggaaccaaacaaga aatctagaat ttggcttgga actttcaaaa cagctgagat 300 ggcagctcgtgctcacgacg tggctgccct agccctccgt ggaagaggcg cctgcctcaa 360 ttatgcggactcggcttggc ggctccgcat cccggagaca acctgccaca aggatatcca 420 gaaggctgctgctgaagccg cattggcttt tgaggctgag aaaagtgatg tgacgatgga 480 ggagacgatggcggtggctt ctcaggctga agtgaatgac acgacgacag atcatggcat 540 gaacatggaggaggcaacag cggtggcttc tcaggctgag gtgaatgaca cgacgacaga 600 tcatggcgtagacatggagg agacgatggt ggaggctgtt tttacggagg aacaaagtga 660 agggttcaacatggcggagg agtcgacggt ggaggctgct gttgttacgg atgaactgag 720 caaaggattttacatggacg aggagtggac gtacgagatg ccgaccttgt tggctgatat 780 ggcggcagggatgcttttgc cgccaccatc tgtacaatgg ggacataatg atgacttgga 840 aggagatgcggacatgaacc tctggagtta ttaatactcg tattttt 887 69 277 PRT Brassicaoleracea boCBF3 polypeptide 69 Met Asn Thr Phe Pro Ala Ser Thr Glu MetVal Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly GlyAsp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala GlyArg Lys Lys Phe Gln Glu Thr 35 40 45 Arg His Pro Ile Tyr Arg Gly Val ArgLeu Arg Lys Ser Gly Lys Trp 50 55 60 Val Cys Glu Val Arg Glu Pro Asn LysLys Ser Arg Ile Trp Leu Gly 65 70 75 80 Thr Phe Lys Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala 85 90 95 Leu Ala Leu Arg Gly Arg Gly AlaCys Leu Asn Tyr Ala Asp Ser Ala 100 105 110 Trp Arg Leu Arg Ile Pro GluThr Thr Cys His Lys Asp Ile Gln Lys 115 120 125 Ala Ala Ala Glu Ala AlaLeu Ala Phe Glu Ala Glu Lys Ser Asp Val 130 135 140 Thr Met Glu Glu ThrMet Ala Val Ala Ser Gln Ala Glu Val Asn Asp 145 150 155 160 Thr Thr ThrAsp His Gly Met Asn Met Glu Glu Ala Thr Ala Val Ala 165 170 175 Ser GlnAla Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met 180 185 190 GluGlu Thr Met Val Glu Ala Val Phe Thr Glu Glu Gln Ser Glu Gly 195 200 205Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val Val Thr Asp 210 215220 Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Tyr Glu Met 225230 235 240 Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu Pro ProPro 245 250 255 Ser Val Gln Trp Gly His Asn Asp Asp Leu Glu Gly Asp AlaAsp Met 260 265 270 Asn Leu Trp Ser Tyr 275 70 950 DNA Brassica oleraceaboCBF4 gene 70 ctgaaaagaa gataaaagag agagaaataa atatcttatc aaaccagacagaacagagat 60 cttgttactt actatactac actcagcctt atccagtttt tcaaaagaagttttcaacta 120 tgaactcagt ctctactttt tctgaacttc ttggctctga gaacgagtctccggtaggtg 180 gtgattactg tcccatgttg gcggcgagct gtccgaagaa gccggcgggtaggaagaagt 240 ttcgggagac acgtcacccc atttaccgag gagttcgcct tagaaaatcaggtaagtggg 300 tgtgtgaagt gagggaacca aacaaaaaat ctaggatttg gctcggaactttcaaaacag 360 ctgagatcgc agctcgtgct cacgacgtcg ccgccttagc tctccgtggaagaggcgcct 420 gcctcaactt cgccgactcg gcttggcggc tccgtatccc ggagacaacctgcgccaagg 480 atatccagaa ggctgctgct gaagccgcat tggcttttga ggccgagaagagtgatacca 540 cgacgaatga tcatggcatg aacatggctt ctcaggctga ggttaatgacacgacggatc 600 atggcctgga catggaggag acgatggtgg aggctgtttt tactgaggagcagagagacg 660 ggttttacat ggcggaggag acgacggtgg agggtgttgt tccggaggaacagatgagca 720 aagggtttta catggacgag gagtggatgt tcgggatgcc gaccttgttggctgatatgg 780 cggcagggat gctcttaccg ccgccgtccg tacaatgggg acataatgatgacttcgaag 840 gagatgctga catgaacctc tggaattatt agtactcgta tttttttaaattattttttg 900 aacgaataat attttattga attcggattc tacctgtttt tttaatggat950 71 250 PRT Brassica oleracea boCBF4 polypeptide 71 Met Asn Ser ValSer Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro ValGly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 20 25 30 Lys Lys ProAla Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 35 40 45 Tyr Arg GlyVal Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 50 55 60 Arg Glu ProAsn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys Thr 65 70 75 80 Ala GluIle Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg 85 90 95 Gly ArgGly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 100 105 110 IlePro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 115 120 125Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn Asp 130 135140 His Gly Met Asn Met Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Asp 145150 155 160 His Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala Val Phe ThrGlu 165 170 175 Glu Gln Arg Asp Gly Phe Tyr Met Ala Glu Glu Thr Thr ValGlu Gly 180 185 190 Val Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr MetAsp Glu Glu 195 200 205 Trp Met Phe Gly Met Pro Thr Leu Leu Ala Asp MetAla Ala Gly Met 210 215 220 Leu Leu Pro Pro Pro Ser Val Gln Trp Gly HisAsn Asp Asp Phe Glu 225 230 235 240 Gly Asp Ala Asp Met Asn Leu Trp AsnTyr 245 250 72 877 DNA Brassica oleracea boCBF5 gene 72 accgctcgagcaacaatgaa cacattccct gcttccactg aaatggttag ctccgagaac 60 gagtctccggttactacggt agtaggaggt gattattatc ccatgttggc ggcaagctgt 120 ccgaagaagccagcgggtag gaagaagttt caggagacac gtcaccccat ttaccgagga 180 gttcgtctgagaaagtcagg taagtgggtg tgtgaagtga gggaactaaa caagaaatct 240 agaatttggcttggaacttt caaaacagct gagatggcag ctcgtgctca cgacgtggct 300 gccctagccctccgtggaag aggcgcctgc ctcaattatg cggactcggc ttggcggctc 360 cgcatcccggagacaacctg ccacaaggat atccagaagg ctgctgctga agccgcattg 420 gcttttgaggctgagaagag tgatgcgacg atgcaaaatg gcctgaacat ggaggagacg 480 acggcggcggcttctcagac tgaagtgagt gacacgacga cagatcatgg catgaacatg 540 gaggagacaacggcggtggc ttctcaggct gaggtgaatg acacgacgac agatcatggc 600 gtagacatggaggagacgat ggtggaggct gtttttactg aggaacaaag tgaagggttc 660 aacatggcgaaggagtcgac ggcggaggct gctgttgtta cggaggaact gagcaaagga 720 gtttacatggacgaggagtg gacgtacgag atgccgacct tgttggctga tatggcggca 780 gggatgcttttgccgccacc atctgtacaa tggggacata atgatgactt ggaaggagat 840 gcggacatgaacctactgga gttattaagg atccgcg 877 73 287 PRT Brassica oleracea boCBF5polypeptide 73 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Ser Ser GluAsn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr ProMet Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys PheGln Glu Thr 35 40 45 Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp 50 55 60 Val Cys Glu Val Arg Glu Leu Asn Lys Lys Ser Arg IleTrp Leu Gly 65 70 75 80 Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala 85 90 95 Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn TyrAla Asp Ser Ala 100 105 110 Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys HisLys Asp Ile Gln Lys 115 120 125 Ala Ala Ala Glu Ala Ala Leu Ala Phe GluAla Glu Lys Ser Asp Ala 130 135 140 Thr Met Gln Asn Gly Leu Asn Met GluGlu Thr Thr Ala Ala Ala Ser 145 150 155 160 Gln Thr Glu Val Ser Asp ThrThr Thr Asp His Gly Met Asn Met Glu 165 170 175 Glu Thr Thr Ala Val AlaSer Gln Ala Glu Val Asn Asp Thr Thr Thr 180 185 190 Asp His Gly Val AspMet Glu Glu Thr Met Val Glu Ala Val Phe Thr 195 200 205 Glu Glu Gln SerGlu Gly Phe Asn Met Ala Lys Glu Ser Thr Ala Glu 210 215 220 Ala Ala ValVal Thr Glu Glu Leu Ser Lys Gly Val Tyr Met Asp Glu 225 230 235 240 GluTrp Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 245 250 255Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Leu 260 265270 Glu Gly Asp Ala Asp Met Asn Leu Leu Glu Leu Leu Arg Ile Arg 275 280285 74 374 DNA Brassica rapa brCBF1 gene 74 catcccattt acaggggggttcgtttaaga aagtcaggta agtgggtgtg tgaagtgagg 60 gaaccaaaca agaaatctaggatttggctc ggaactttca aaaccgctga gatcgctgct 120 cgtgctcacg acgttgctgccttagccctc cgcgggagag gcgcctgcct caacttcgcc 180 gactcggctt ggcggctccgtatcccggag acaacctgcg ccaaggacat ccagaaggcg 240 gctgctgaag ctgcattggcttttgaggcc gagaagagtg atcatggcat gaacatcaag 300 aatactacgg cggtggtttctcaggttgag gtgaatgaca cgacgacgga ccacggcttg 360 gacatggagg agac 374 75124 PRT Brassica rapa brCBF1 polypeptide 75 His Pro Ile Tyr Arg Gly ValArg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu ProAsn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu IleAla Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg GlyAla Cys Leu Asn Phe Ala Asp Ser Ala Trp 50 55 60 Arg Leu Arg Ile Pro GluThr Thr Cys Ala Lys Asp Ile Gln Lys Ala 65 70 75 80 Ala Ala Glu Ala AlaLeu Ala Phe Glu Ala Glu Lys Ser Asp His Gly 85 90 95 Met Asn Ile Lys AsnThr Thr Ala Val Val Ser Gln Val Glu Val Asn 100 105 110 Asp Thr Thr ThrAsp His Gly Leu Asp Met Glu Glu 115 120 76 884 DNA Brassica rapa brCBF2gene 76 tacactcagc cttatccagt ttttttcaaa agacttttca acaatgaacacattccctgc 60 gtccactgaa atggttggct ccgagaacga gtctccggtt actacggtagcaggaggtga 120 ttattatccc atgttggcgg caagctgtcc gaagaagcca gcgggtaggaagaagtttca 180 ggagacacgt caccccattt accgaggagt tcgtctgaga aagtcaggtaagtgggtgtg 240 tgaagtgagg gaaccaaaca agaaatctag aatttggctt ggaactttcaaaacagctga 300 gatggcagct cgtgctcacg acgtcgctgc cctagccctc cgtggaagaggcgcctgcct 360 caattatgcg gactcggctt ggcggctccg catcccggag acaacctgccacaaggatat 420 ccagaaggct gctgctgaag ccgcattggc ttttgaggct gagaaaagtgatgtgacgat 480 gcaaaatggc ctgaacatgg aggagatgac ggcggtggct tctcaggctgaagtgaatga 540 cacgacgaca gaacatggca tgaacatgga ggaggcaacg gcagtggcttctcaggctga 600 ggtgaatgac acgacgacgg atcatggcgt agacatggag gagacaatggtggaggctgt 660 ttttactgag gaacaaagtg aagggtttaa catggcgaag gagtcgacggtggaggctgc 720 tgttgttacg gaggaaccga gcaaaggatc ttacatggac gaggagtggatgctcgagat 780 gccgaccttg ttggctgata tggcggaagg gatgcttttg ccgccgccgtccgtacaatg 840 gggacagaat gatgacttcg aaggagatgc tgacatgaac ctct 884 77280 PRT Brassica rapa brCBF2 polypeptide 77 Met Asn Thr Phe Pro Ala SerThr Glu Met Val Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr ValAla Gly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys LysPro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp 50 55 60 Val Cys Glu Val Arg GluPro Asn Lys Lys Ser Arg Ile Trp Leu Gly 65 70 75 80 Thr Phe Lys Thr AlaGlu Met Ala Ala Arg Ala His Asp Val Ala Ala 85 90 95 Leu Ala Leu Arg GlyArg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 100 105 110 Trp Arg Leu ArgIle Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 115 120 125 Ala Ala AlaGlu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 130 135 140 Thr MetGln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala Ser 145 150 155 160Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 165 170175 Glu Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 180185 190 Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr195 200 205 Glu Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr ValGlu 210 215 220 Ala Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr MetAsp Glu 225 230 235 240 Glu Trp Met Leu Glu Met Pro Thr Leu Leu Ala AspMet Ala Glu Gly 245 250 255 Met Leu Leu Pro Pro Pro Ser Val Gln Trp GlyGln Asn Asp Asp Phe 260 265 270 Glu Gly Asp Ala Asp Met Asn Leu 275 28078 806 DNA Brassica rapa brCBF3 gene 78 acactcagcc ttatccagtt ttcaaaaaaagtattcaacg atgaactcag tctctacttt 60 ttctgaactg ctctgctccg agaacgagtctccggttaat acggaaggtg gtgattacat 120 tttggcggcg agctgtccca agaaacctgctggtaggaag aagtttcagg agacacgcca 180 ccccatttac agaggagttc gtctgaggaagtcaggtaag tgggtgtgtg aagtgaggga 240 accaaacaag aaatctagaa tttggctcggaactttcaaa acagctgaga tcgcagctcg 300 tgctcacgac gttgccgcct tagctctccgtggaagaggc gcctgcctca acttcgccga 360 ctcggcttgg cggctccgta tcccggagacgacctgcgcc aaggatatcc agaaggctgc 420 tgctgaagcc gcattggctt ttgaggccgagaagagtgat accacgacga atgatcgtgg 480 catgaacatg gaggagacgt cggcggtggcttctccggct gagttgaatg atacgacggc 540 ggatcatggc ctggacatgg aggagacgatggtggaggct gtttttaggg aggaacagag 600 agaagggttt tacatggcgg aggagacgacggtggagggt gttgttccgg agtaacagat 660 gagcaaaggg ttttacatgg acgaggagtggacgttcgag atgccgaggt tgttggctga 720 tatggcggaa gggatgcttt tgccgcccccgtccgtacaa tggggacata acgatgactt 780 cgaaggagat gctgacatga acctct 806 79204 PRT Brassica rapa brCBF3 polypeptide 79 Met Asn Ser Val Ser Thr PheSer Glu Leu Leu Cys Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Asn Thr GluGly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 20 25 30 Pro Lys Lys Pro Ala GlyArg Lys Lys Phe Gln Glu Thr Arg His Pro 35 40 45 Ile Tyr Arg Gly Val ArgLeu Arg Lys Ser Gly Lys Trp Val Cys Glu 50 55 60 Val Arg Glu Pro Asn LysLys Ser Arg Ile Trp Leu Gly Thr Phe Lys 65 70 75 80 Thr Ala Glu Ile AlaAla Arg Ala His Asp Val Ala Ala Leu Ala Leu 85 90 95 Arg Gly Arg Gly AlaCys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 100 105 110 Arg Ile Pro GluThr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 115 120 125 Glu Ala AlaLeu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn 130 135 140 Asp ArgGly Met Asn Met Glu Glu Thr Ser Ala Val Ala Ser Pro Ala 145 150 155 160Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Met Glu Glu Thr 165 170175 Met Val Glu Ala Val Phe Arg Glu Glu Gln Arg Glu Gly Phe Tyr Met 180185 190 Ala Glu Glu Thr Thr Val Glu Gly Val Val Pro Glu 195 200 80 755DNA Brassica rapa brCBF4 gene 80 accgctcgag tacttactat actacactcagccttatcca gtttttcttc caacgatgga 60 ctcaatctct acttttcctg aactgcttggctcagagaac gagtctccgg ttactacggt 120 agtaggaggt gattattgtc ccaggttggcggcaagctgt ccgaagaagc cagcgggtag 180 gaagaagttt caggagacac gtcaccccatttaccgtgga gttcgtttaa gaaagtccgg 240 taagtgggtg tgtgaagtga gggaaccaaacaagaaatct aggatttggc tcggaacttt 300 caaaaccgct gagatcgctg ctcgtgctcacgacgttgct gccttagccc tccgcggaag 360 aggcgcctgc ctcaacttcg ccgactcggcttgacggctc cgtatcccgg agacaacctg 420 cgccaaggat atccagaagg ctgctgctgaagctgcattg gcttttgagg ccgagaagag 480 tgatcatggc atgaacatga agaatactacggcggtggct tctcaggttg aggtgaatga 540 tacgacgacg gaccatggcg tggacatggaggagacgagg gtggagggtg ttgttacgga 600 ggaacagaac aattggtttt acatggacgaggagtggatg tttgggatgc cgacgttgtt 660 ggttgatatg gcggaaggga tgcttataccgcggcagtcc gtacaatcgg gacactacga 720 tgacttcgaa ggagatgctg acatgaacctctgga 755 81 112 PRT Brassica rapa brCBF4 polypeptide 81 Met Asp Ser IleSer Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro ValThr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 20 25 30 Ala Ser CysPro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg His ProIle Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 50 55 60 Val Cys GluVal Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 65 70 75 80 Thr PheLys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala 85 90 95 Leu AlaLeu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala 100 105 110 82832 DNA Brassica rapa brCBF5 gene 82 accgctcgag tacttactat actacactcagccttatcca gtttttcttc caacgatgga 60 ctcaatctct acttttcctg aactgcttggctcagagaac gagtctccgg ttactacggt 120 agtaggaggt gattattgtc ccaggttggcggcaagctgt ccgaagaagc cagcgggtag 180 gaagaagttt caggagacac gtcaccccatttaccgtgga gttcgtttaa gaaagtccgg 240 taagtgggtg tgtgaagtga gggaaccaaacaagaaatct aggatttggc tcggaacttt 300 caaaaccgct gagatcgctg ctcgtgctcacgacgttgct gccttagccc tccgcggaag 360 aggcgcctgc ctcaacttcg ccgactcggcttggcggctc cgtatcccgg agacaacctg 420 cgccaaggat atccagaagg ctgctgctgaagctgctttg gcttttgagg ccgagaagag 480 tgatcatggc atgaacatga agaatactacggcggtggct tctcaggttg aggtgaatga 540 tacgacgacg gaccatggcg tggacatggaggagacgttg gtggaggctg tttttacgga 600 ggaacagaga gaagggtttt acatgacggaggagacgagg gtggagggtg ttgttacgga 660 ggaacagaac aattggtttt acatggacgaggagtggatg tttgggatgc cgacgttgtt 720 ggttgatatg gcggaaggga tgcttataccgcggcagtcc gtacaatcgg gacactacga 780 tgacttcgaa ggagatgctg acatgaacctctggaattat tagggatccg cg 832 83 255 PRT Brassica rapa brCBF5 polypeptide83 Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 1 510 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 2025 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 3540 45 Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 5055 60 Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 6570 75 80 Thr Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala85 90 95 Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala100 105 110 Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile GlnLys 115 120 125 Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys SerAsp His 130 135 140 Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser GlnVal Glu Val 145 150 155 160 Asn Asp Thr Thr Thr Asp His Gly Val Asp MetGlu Glu Thr Leu Val 165 170 175 Glu Ala Val Phe Thr Glu Glu Gln Arg GluGly Phe Tyr Met Thr Glu 180 185 190 Glu Thr Arg Val Glu Gly Val Val ThrGlu Glu Gln Asn Asn Trp Phe 195 200 205 Tyr Met Asp Glu Glu Trp Met PheGly Met Pro Thr Leu Leu Val Asp 210 215 220 Met Ala Glu Gly Met Leu IlePro Arg Gln Ser Val Gln Ser Gly His 225 230 235 240 Tyr Asp Asp Phe GluGly Asp Ala Asp Met Asn Leu Trp Asn Tyr 245 250 255 84 830 DNA Brassicarapa brCBF6 gene 84 tactacactc agccttatcc agttttcaaa aaaagtattcaactatgaac tcagtctcta 60 ctttttctga actgctctgc tccgagaaca agtctccggttaatacggaa ggtggtgatt 120 acattttggc ggcgagctgt cccaagaaac ctgctggtaggaagaagttt caggagacac 180 gccaccccat ttacagagga gttcgcctaa gaaagtcaggtaagtgggtg tgtgaagtga 240 gggaaccaaa caagaaatct agaatttggc tcggaactttcaaaacagct gagatagcag 300 ctcgtgctca cgacgtcgcc gccttagctc tccgtggaagaggcgcctgc ctcaacttcg 360 ccgactcggc ttggcggctc cgtatcccag agacaacctgcgccaaggat atccagaagg 420 ctgctgctga agccgcattg gcttttgagg ccgagaagagtgataccacg acgaatgatc 480 gtggcatgaa catggaggag acgtccgcgg tggcttctccggctgagttg aatgatacga 540 cggcggatca tggcctggac atggaggaga cgatggtggaggctgttttt agggacgaac 600 agagagaagg gttttacatg gcggaggaga cgacggtggagggtgttgtt ccggaggaac 660 agatgagcaa agggttttac atggacgagg agtggacgttcgagatgccg aggttgttgg 720 ctgatatggc ggaagggatg cttctgcctc ccccgtccgtacaatgggga cataacgatg 780 acttcgaagg agatgctgac atgaacctct ggaattattagggatccgcg 830 85 258 PRT Brassica rapa brCBF6- polypeptide 85 Met AsnSer Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Lys 1 5 10 15 SerPro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 20 25 30 ProLys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg His Pro 35 40 45 IleTyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu 50 55 60 ValArg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys 65 70 75 80Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 85 90 95Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 100 105110 Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 115120 125 Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn130 135 140 Asp Arg Gly Met Asn Met Glu Glu Thr Ser Ala Val Ala Ser ProAla 145 150 155 160 Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp MetGlu Glu Thr 165 170 175 Met Val Glu Ala Val Phe Arg Asp Glu Gln Arg GluGly Phe Tyr Met 180 185 190 Ala Glu Glu Thr Thr Val Glu Gly Val Val ProGlu Glu Gln Met Ser 195 200 205 Lys Gly Phe Tyr Met Asp Glu Glu Trp ThrPhe Glu Met Pro Arg Leu 210 215 220 Leu Ala Asp Met Ala Glu Gly Met LeuLeu Pro Pro Pro Ser Val Gln 225 230 235 240 Trp Gly His Asn Asp Asp PheGlu Gly Asp Ala Asp Met Asn Leu Trp 245 250 255 Asn Tyr 86 854 DNABrassica rapa brCBF7 gene 86 ctatactaca cacagcctta tccagccgct cgagtacttactatactaca ctcagccttt 60 tccagttttt caaaagaagt tttcaacgat gaactcagtctctactcttt ctgaagttct 120 tggctcccag aacgagtctc ccgtaggtgg tgattactgtcccatgttgg cggcgagctg 180 tccgaagaag ccggcgggta ggaagaagtt tcgggagacacgtcacccca tttacagagg 240 agttcgtctt agaaagtcag gtaagtgggt gtgtgaagtgagggaaccaa acaagaaatc 300 taggatttgg ctcggaactt tcaaaacagc tgagatcgcagctcgtgctc acgacgttgc 360 cgccttagct ctccgtggaa gaggcgcctg cctcaacttcgccgactcgg cttggcggct 420 ccgtatcccg gagacaacct gcgccaagga tatccagaaggctgctgctg aagccgcatt 480 ggcttttgag gcggagaaga gtgataccac gacgacgaatgatcatggca tgaacatggc 540 ttctcaggtt gaggttaatg acacgacgga tcatgacctggacatggagg agacgatggt 600 ggaggctgtt tttagggagg aacagagaga agggttttacatggcggagg agacgacggt 660 ggagggtatt gttccggagg aacagatgag caaagggttttacatggacg aggagtggat 720 gttcgggatg ccgaccttgt tggctgatat ggcggcagggatgctcttac cgccgccgtc 780 cgtacaatgg ggacataatg atgacttcga aggagatgctgacatgaacc tctggaatta 840 ttaagggatc cgcg 854 87 251 PRT Brassica rapabrCBF7 polypeptide 87 Met Asn Ser Val Ser Thr Leu Ser Glu Val Leu GlySer Gln Asn Glu 1 5 10 15 Ser Pro Val Gly Gly Asp Tyr Cys Pro Met LeuAla Ala Ser Cys Pro 20 25 30 Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg GluThr Arg His Pro Ile 35 40 45 Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly LysTrp Val Cys Glu Val 50 55 60 Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp LeuGly Thr Phe Lys Thr 65 70 75 80 Ala Glu Ile Ala Ala Arg Ala His Asp ValAla Ala Leu Ala Leu Arg 85 90 95 Gly Arg Gly Ala Cys Leu Asn Phe Ala AspSer Ala Trp Arg Leu Arg 100 105 110 Ile Pro Glu Thr Thr Cys Ala Lys AspIle Gln Lys Ala Ala Ala Glu 115 120 125 Ala Ala Leu Ala Phe Glu Ala GluLys Ser Asp Thr Thr Thr Thr Asn 130 135 140 Asp His Gly Met Asn Met AlaSer Gln Val Glu Val Asn Asp Thr Thr 145 150 155 160 Asp His Asp Leu AspMet Glu Glu Thr Met Val Glu Ala Val Phe Arg 165 170 175 Glu Glu Gln ArgGlu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu 180 185 190 Gly Ile ValPro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu 195 200 205 Glu TrpMet Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 210 215 220 MetLeu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe 225 230 235240 Glu Gly Asp Ala Asp Met Asn Leu Trp Asn Tyr 245 250 88 738 DNAGlycine max gmCBF1 gene 88 catccgattt atagtggcgt gaggaggagg aacacggataagtgggtaag tgaggtgagg 60 gagcccaaca aaaagaccag gatttggctg gggacttttcccacgccgga gatggcggca 120 cgggcccacg acgtggcggc aatggccctg aggggccggtatgcctgtct caacttcgct 180 gactcgacgt ggcggttacc aattcccgcc actgctaacgcaaaggatat acagaaagca 240 gcagcagagg ctgccgaggc tttcagacca agtcagaccttagaaaatac gaatacaaag 300 caagagtgtg taaaagtggt gacgacaaca acgatcacagaacaaaaacg aggaatgttt 360 tatacggagg aagaagagca agtgttagat atgcctgagttgcttaggaa tatggtgctt 420 atgtccccaa cacattgcat agggtatgag tatgaagatgctgacttgga tgctcaagat 480 gctgaggtgt ccctatggag tttctcaatt taataacgtgcttttggttt ggttttttat 540 gttagttttg gagtgtgact gtctgtactg gttttttattagtagtacgg atactagcta 600 taggtggcag attgaaaggg accaaaagga attttcttttgaaacccttt ttgtcaaagt 660 aatcaatcgc gtatcatcaa gtgaatccct tgatcaagtttatgtatgaa ttaaataaaa 720 gaagaatcta gttttggt 738 89 170 PRT Glycine maxgmCBF1 polypeptide 89 His Pro Ile Tyr Ser Gly Val Arg Arg Arg Asn ThrAsp Lys Trp Val 1 5 10 15 Ser Glu Val Arg Glu Pro Asn Lys Lys Thr ArgIle Trp Leu Gly Thr 20 25 30 Phe Pro Thr Pro Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Met 35 40 45 Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn PheAla Asp Ser Thr Trp 50 55 60 Arg Leu Pro Ile Pro Ala Thr Ala Asn Ala LysAsp Ile Gln Lys Ala 65 70 75 80 Ala Ala Glu Ala Ala Glu Ala Phe Arg ProSer Gln Thr Leu Glu Asn 85 90 95 Thr Asn Thr Lys Gln Glu Cys Val Lys ValVal Thr Thr Thr Thr Ile 100 105 110 Thr Glu Gln Lys Arg Gly Met Phe TyrThr Glu Glu Glu Glu Gln Val 115 120 125 Leu Asp Met Pro Glu Leu Leu ArgAsn Met Val Leu Met Ser Pro Thr 130 135 140 His Cys Ile Gly Tyr Glu TyrGlu Asp Ala Asp Leu Asp Ala Gln Asp 145 150 155 160 Ala Glu Val Ser LeuTrp Ser Phe Ser Ile 165 170 90 793 DNA Raphanus sativus rsCBF1 gene 90actacactca gccttatcca gtttttcttc caacgatgga ctcaatctct actttttctg 60aactgcttgg ctccgagaac gagtctccgg ttactacggt agtaggaggt gattattttc 120ccaggttggc ggcaagctgt ccgaagaagc cagcgggtag gaagaagttt caggagacac 180gtcaccccat ttaccgcgga gttcgtttaa gaaagtcagg taagtgggtg tgtgaagtga 240gggaaccaaa caagaaatct aggatttggc tcggaacttt caaaaccgct gagatcgctg 300ctcgtgctca cgacgttgct gccttagccc tccgcggaag aggcgcctgc ctcaacttcg 360ccgactcggc ttggcggctc cgtatcccgg agacaacctg cgccaaggat atccagaagg 420ctgctgctga agctgcattg gcttttgagg ccgagaagag tgatcatggc atgaacatga 480agaatactac ggcggtggct tctcaggttg aggtgaatga cacgacgacg gaccatggcg 540tggacatgga ggagacgttg gtggaggctg tttttacgga ggaacagaga gaagggtttt 600acatgacgga ggagacgagg gtggagggtg ttgttacgga ggaacagaac aattggtttt 660acatggacga ggagtggatg tttgggatgc cgacgttgtt ggttgatatg gcggaaggga 720tgcttttacc gcggccgtcc gtacaatcgg gacactacga tgacttcgaa ggagatgctg 780acatgaacct ctg 793 91 252 PRT Raphanus sativus rsCBF1 polypeptide 91 MetAsp Ser Ile Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 1 5 10 15Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Phe Pro Arg Leu Ala 20 25 30Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 50 55 60Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 65 70 7580 Thr Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala 85 9095 Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala 100105 110 Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys115 120 125 Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser AspHis 130 135 140 Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser Gln ValGlu Val 145 150 155 160 Asn Asp Thr Thr Thr Asp His Gly Val Asp Met GluGlu Thr Leu Val 165 170 175 Glu Ala Val Phe Thr Glu Glu Gln Arg Glu GlyPhe Tyr Met Thr Glu 180 185 190 Glu Thr Arg Val Glu Gly Val Val Thr GluGlu Gln Asn Asn Trp Phe 195 200 205 Tyr Met Asp Glu Glu Trp Met Phe GlyMet Pro Thr Leu Leu Val Asp 210 215 220 Met Ala Glu Gly Met Leu Leu ProArg Pro Ser Val Gln Ser Gly His 225 230 235 240 Tyr Asp Asp Phe Glu GlyAsp Ala Asp Met Asn Leu 245 250 92 682 DNA Raphanus sativus rsCBF2 gene92 acacctaaac cttatccagg tttaactttt tttttcataa agagttttca acaatgacca 60cattttctac cttttccgaa atgttgggct ccgagtacga gtctccggtt acattaggcg 120gagagtattg tccgaagctg gccgcgagct gtccgaagaa accagctggt cgtaagaagt 180ttcgagagac gcgccaccca atatacagag gagttcgtct gagaaactca ggtaagtggg 240tgtgtgaagt gagggagcca aacaagaaat ctaggatttg gctcggtact ttcctaaccg 300ccgagatcgc agcgcgtgcc cacgacgtcg ccgccatagc cctccgcggc aaatccgcat 360gtctcaattt cgctgactcg gcttggcggc tccgtatccc ggagacaaca tgccccaagg 420atatacagaa ggcggctgct gaagccgcgg tggcttttca ggctgagata aatgatacga 480cgacggatca tggcctggac ttggaggaga cgatcgtgga ggctattttt acggaggtaa 540acaacgatga gttttatatg gacgaggagt ccatgttcgg gatgccgtct ttgttggcta 600gtatggcgga agggatgctt ttgccgctgc cgtccgtaca atctgaacat aactgtgact 660tcgacggaga tgctgacatg aa 682 93 209 PRT Raphanus sativus rsCBF2-polypeptide 93 Met Thr Thr Phe Ser Thr Phe Ser Glu Met Leu Gly Ser GluTyr Glu 1 5 10 15 Ser Pro Val Thr Leu Gly Gly Glu Tyr Cys Pro Lys LeuAla Ala Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg GluThr Arg His 35 40 45 Pro Ile Tyr Arg Gly Val Arg Leu Arg Asn Ser Gly LysTrp Val Cys 50 55 60 Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp LeuGly Thr Phe 65 70 75 80 Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp ValAla Ala Ile Ala 85 90 95 Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala AspSer Ala Trp Arg 100 105 110 Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys AspIle Gln Lys Ala Ala 115 120 125 Ala Glu Ala Ala Val Ala Phe Gln Ala GluIle Asn Asp Thr Thr Thr 130 135 140 Asp His Gly Leu Asp Leu Glu Glu ThrIle Val Glu Ala Ile Phe Thr 145 150 155 160 Glu Val Asn Asn Asp Glu PheTyr Met Asp Glu Glu Ser Met Phe Gly 165 170 175 Met Pro Ser Leu Leu AlaSer Met Ala Glu Gly Met Leu Leu Pro Leu 180 185 190 Pro Ser Val Gln SerGlu His Asn Cys Asp Phe Asp Gly Asp Ala Asp 195 200 205 Met 94 349 DNAZea mays zmCBF1 gene 94 cggagtccgc ggacggcggc ggcggcggcg acgacgagtacgcgacggtg ctgtcggcgc 60 cacccaagcg gccggcgggg cggaccaagt tccgggagacgcggcacccc gtgtaccgcg 120 gcgtgcggcg gcgcgggccc gcggggcgct gggtgtgcgaggtccgcgag cccaacaaga 180 agtcgcgcat ctggctcggc accttcgcca cccccgaggccgccgcgcgc gcgcacgacg 240 tggccgcgct ggccctgcgg ggccgcgccg cgtgcctcaacttcgccgac tcggcgcgcc 300 tgctccaagt cgaccccgcc acgctcgcca cccccgacgacatccgccg 349 95 115 PRT Zea mays zmCBF1- polypeptide 95 Glu Ser Ala AspGly Gly Gly Gly Gly Asp Asp Glu Tyr Ala Thr Val 1 5 10 15 Leu Ser AlaPro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu 20 25 30 Thr Arg HisPro Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly 35 40 45 Arg Trp ValCys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp 50 55 60 Leu Gly ThrPhe Ala Thr Pro Glu Ala Ala Ala Arg Ala His Asp Val 65 70 75 80 Ala AlaLeu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp 85 90 95 Ser AlaArg Leu Leu Gln Val Asp Pro Ala Thr Leu Ala Thr Pro Asp 100 105 110 AspIle Arg 115 96 675 DNA Arabidopsis thaliana G912 gene 96 ttactcgtcaaaactccaga gtgacacgtc acccactccg tcaaagtcgt tatgattcca 60 gccaacttccggcggcggca aaagcatccc ctccgccata ttctcaaaaa agttgggcat 120 ccccaaaagcgcctcatcat ccatataaaa cacaccacca ttctgctcct ccgccctcct 180 ctccccctccctcaccccct cccctgccgc ctcctctgcc tccgccgcag ttttagatcc 240 ctccgtcgtagtctcattct gaaacgccat tgcagcttca gacgcagctt tctgaatctc 300 cttaggacaagtagtctcag gaatacgaag ccgccaagca gaatcagcga aattgagaca 360 agcagagcgaccacgaagag ctaaagcagc aacatcatga gcacgagcag ccatttcaac 420 cgtcggaaaagtacctaacc aaatcctaga tttcttatta ggctctctaa cttcacaaac 480 ccatttaccagaattcctct gacgaactcc tctgtaaatc ggatgacgtg tctcacgaaa 540 cttcttcctcccagctcgtt tctttggaca acttgaagct aactttggtg aacactcact 600 actgtctgaaaccggagatc tatgatcgga gattgagaga aacgagtctg ggaatgtaga 660 gtaaaatggattcat 675 97 224 PRT Arabidopsis thaliana G912 polypeptide 97 Met AsnPro Phe Tyr Ser Thr Phe Pro Asp Ser Phe Leu Ser Ile Ser 1 5 10 15 AspHis Arg Ser Pro Val Ser Asp Ser Ser Glu Cys Ser Pro Lys Leu 20 25 30 AlaSer Ser Cys Pro Lys Lys Arg Ala Gly Arg Lys Lys Phe Arg Glu 35 40 45 ThrArg His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys 50 55 60 TrpVal Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu 65 70 75 80Gly Thr Phe Pro Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala 85 90 95Ala Leu Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 100 105110 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 115120 125 Lys Ala Ala Ser Glu Ala Ala Met Ala Phe Gln Asn Glu Thr Thr Thr130 135 140 Glu Gly Ser Lys Thr Ala Ala Glu Ala Glu Glu Ala Ala Gly GluGly 145 150 155 160 Val Arg Glu Gly Glu Arg Arg Ala Glu Glu Gln Asn GlyGly Val Phe 165 170 175 Tyr Met Asp Asp Glu Ala Leu Leu Gly Met Pro AsnPhe Phe Glu Asn 180 185 190 Met Ala Glu Gly Met Leu Leu Pro Pro Pro GluVal Gly Trp Asn His 195 200 205 Asn Asp Phe Asp Gly Val Gly Asp Val SerLeu Trp Ser Phe Asp Glu 210 215 220 98 630 DNA Arabidopsis thalianaG2513 gene 98 atgaataatg atgatattat tctggcggag atgaggccta agaagcgtgcgggaaggaga 60 gtgtttaagg agacacgtca cccagtttac agaggcataa ggcggaggaacggtgacaaa 120 tgggtctgcg aagtcagaga accgacgcac caacgccgca tttggctcgggacttatccc 180 acagcagata tggcagcgcg tgcacacgac gtggcggttt tagctctgcgtgggagatcc 240 gcatgtttga atttcgccga ctccgcttgg cggcttccgg tgccggaatccaatgatccg 300 gatgtgataa gaagagttgc ggcggaagct gcggagatgt ttaggccggtggatttagaa 360 agtggaatta cggttttgcc ttgtgcggga gatgatgtgg atttgggttttggttcgggt 420 tccggctctg gttcgggatc ggaggagagg aattcttctt cgtatggatttggagactac 480 gaagaagtct caacgacgat gatgagactc gcggaggggc cactaatgtcgccgccgcga 540 tcgtatatgg aagacatgac tcctactaat gtttacacgg aagaagagatgtgttatgaa 600 gatatgtcat tgtggagtta cagatattaa 630 99 209 PRTArabidopsis thaliana G2513 polypeptide 99 Met Asn Asn Asp Asp Ile IleLeu Ala Glu Met Arg Pro Lys Lys Arg 1 5 10 15 Ala Gly Arg Arg Val PheLys Glu Thr Arg His Pro Val Tyr Arg Gly 20 25 30 Ile Arg Arg Arg Asn GlyAsp Lys Trp Val Cys Glu Val Arg Glu Pro 35 40 45 Thr His Gln Arg Arg IleTrp Leu Gly Thr Tyr Pro Thr Ala Asp Met 50 55 60 Ala Ala Arg Ala His AspVal Ala Val Leu Ala Leu Arg Gly Arg Ser 65 70 75 80 Ala Cys Leu Asn PheAla Asp Ser Ala Trp Arg Leu Pro Val Pro Glu 85 90 95 Ser Asn Asp Pro AspVal Ile Arg Arg Val Ala Ala Glu Ala Ala Glu 100 105 110 Met Phe Arg ProVal Asp Leu Glu Ser Gly Ile Thr Val Leu Pro Cys 115 120 125 Ala Gly AspAsp Val Asp Leu Gly Phe Gly Ser Gly Ser Gly Ser Gly 130 135 140 Ser GlySer Glu Glu Arg Asn Ser Ser Ser Tyr Gly Phe Gly Asp Tyr 145 150 155 160Glu Glu Val Ser Thr Thr Met Met Arg Leu Ala Glu Gly Pro Leu Met 165 170175 Ser Pro Pro Arg Ser Tyr Met Glu Asp Met Thr Pro Thr Asn Val Tyr 180185 190 Thr Glu Glu Glu Met Cys Tyr Glu Asp Met Ser Leu Trp Ser Tyr Arg195 200 205 Tyr 100 546 DNA Arabidopsis thaliana G2107 gene 100ttagtaactc caaagtgaca aatcttcgta acacatttct tcgtccacgt acacactcgt 60attcatatca atgtacgatc ttggcggcga catcaacggc tcctccgcga gcctcatcat 120cattccagcg actccttcat ccgacgtgtc aaactcactg gctgagggta aaaccgtaat 180tcctgtacta aactccggcg gcctgaacat ctccgctgct tcggccgccg tgcgcctgat 240cgtgtccgga tcagtggatg ccggcaccgg caacctccaa gcagaatcgg agaaattcaa 300acacgcggat ctcccgcgca gagcaagaac cgccacgtcg tgagcacgtg cggccatatc 360tgccgtcgga taagttccga gccagactcg acgctgatga atcggttcac ggacttcgca 420tacccatttg tcgccgtccc tacgccgcac gcctctgtag attgggtgac gtgtctcctt 480gaaaatcctc cgtccagcac gcttctttgg cttcatctcc gccacggtga tatcgtcgtt 540ttccat 546 101 181 PRT Arabidopsis thaliana G2107 polypeptide 101 MetGlu Asn Asp Asp Ile Thr Val Ala Glu Met Lys Pro Lys Lys Arg 1 5 10 15Ala Gly Arg Arg Ile Phe Lys Glu Thr Arg His Pro Ile Tyr Arg Gly 20 25 30Val Arg Arg Arg Asp Gly Asp Lys Trp Val Cys Glu Val Arg Glu Pro 35 40 45Ile His Gln Arg Arg Val Trp Leu Gly Thr Tyr Pro Thr Ala Asp Met 50 55 60Ala Ala Arg Ala His Asp Val Ala Val Leu Ala Leu Arg Gly Arg Ser 65 70 7580 Ala Cys Leu Asn Phe Ser Asp Ser Ala Trp Arg Leu Pro Val Pro Ala 85 9095 Ser Thr Asp Pro Asp Thr Ile Arg Arg Thr Ala Ala Glu Ala Ala Glu 100105 110 Met Phe Arg Pro Pro Glu Phe Ser Thr Gly Ile Thr Val Leu Pro Ser115 120 125 Ala Ser Glu Phe Asp Thr Ser Asp Glu Gly Val Ala Gly Met MetMet 130 135 140 Arg Leu Ala Glu Glu Pro Leu Met Ser Pro Pro Arg Ser TyrIle Asp 145 150 155 160 Met Asn Thr Ser Val Tyr Val Asp Glu Glu Met CysTyr Glu Asp Leu 165 170 175 Ser Leu Trp Ser Tyr 180 102 888 DNAArabidopsis thaliana G21 102 tcattcagat agaaaaaacg gctcttcaag ccgaaacccagcatcggctc cacaaagctg 60 ccacgtggac gagtagtagc aaaacgcatc gtttcgtatcatcatctcat tctcatcggt 120 aaacaaatcc ggcaaatcaa acagcttctc ttcctcactgtctttgtccg tacacgccga 180 agtcgaagca cacgaagctt ccgaatactc ttgactctgagtcgtcgtcg tcgtgcttgt 240 gtccgaagaa aacaactgag ccaccacggc tcgactcggctcggcttcaa ctatttcagc 300 cacttcagaa ttactcacat cgttgaccga atcttgccagttaacggccg ctaaagaggc 360 ggcggcttga atgtctttag gagaatttgt gactggacgaggaagctcgc cggctaactt 420 gggaaaattg aggtaagccg ttgtaccttt aatggctaaagccgctacgt catgagctcg 480 agctgccatc tcagccgttg gataagtccc gagccagattcttgatttct ttctcggctc 540 tctaatctcc gacacccatt ttccccaact cctcatcctcactcctctat acgtcggatg 600 tttatctcca ccgttggtct ttcgccgttt cccacctccaccgttttgat catcatcgtc 660 ttcaacggag actaacgatg acaatgatct tcgagatgcttttcttttag agttgtcttc 720 ctcaatgaag ttagaatttg ttgatgacga agaagaggaagatgtagtag gtgaagagga 780 caatgaggcg gaagcggtga tggaggagga ggaagatacggcggggatgg cggaggagat 840 aaaggtaact tgagaaacac tactctctat gttgatttgtcttgccat 888 103 295 PRT Arabidopsis thaliana G21 polypeptide 103 MetAla Arg Gln Ile Asn Ile Glu Ser Ser Val Ser Gln Val Thr Phe 1 5 10 15Ile Ser Ser Ala Ile Pro Ala Val Ser Ser Ser Ser Ser Ile Thr Ala 20 25 30Ser Ala Ser Leu Ser Ser Ser Pro Thr Thr Ser Ser Ser Ser Ser Ser 35 40 45Ser Thr Asn Ser Asn Phe Ile Glu Glu Asp Asn Ser Lys Arg Lys Ala 50 55 60Ser Arg Arg Ser Leu Ser Ser Leu Val Ser Val Glu Asp Asp Asp Asp 65 70 7580 Gln Asn Gly Gly Gly Gly Lys Arg Arg Lys Thr Asn Gly Gly Asp Lys 85 9095 His Pro Thr Tyr Arg Gly Val Arg Met Arg Ser Trp Gly Lys Trp Val 100105 110 Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser Arg Ile Trp Leu Gly Thr115 120 125 Tyr Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala AlaLeu 130 135 140 Ala Ile Lys Gly Thr Thr Ala Tyr Leu Asn Phe Pro Lys LeuAla Gly 145 150 155 160 Glu Leu Pro Arg Pro Val Thr Asn Ser Pro Lys AspIle Gln Ala Ala 165 170 175 Ala Ser Leu Ala Ala Val Asn Trp Gln Asp SerVal Asn Asp Val Ser 180 185 190 Asn Ser Glu Val Ala Glu Ile Val Glu AlaGlu Pro Ser Arg Ala Val 195 200 205 Val Ala Gln Leu Phe Ser Ser Asp ThrSer Thr Thr Thr Thr Thr Gln 210 215 220 Ser Gln Glu Tyr Ser Glu Ala SerCys Ala Ser Thr Ser Ala Cys Thr 225 230 235 240 Asp Lys Asp Ser Glu GluGlu Lys Leu Phe Asp Leu Pro Asp Leu Phe 245 250 255 Thr Asp Glu Asn GluMet Met Ile Arg Asn Asp Ala Phe Cys Tyr Tyr 260 265 270 Ser Ser Thr TrpGln Leu Cys Gly Ala Asp Ala Gly Phe Arg Leu Glu 275 280 285 Glu Pro PhePhe Leu Ser Glu 290 295 104 20 DNA Artificial Sequence PCR primer O368104 cayccnatht aymgnggngt 20 105 26 DNA Artificial Sequence PCR primerO376 105 gcngcytcng cngcngcytt ytgdat 26 106 24 DNA Artificial SequencePCR primer O2953 106 aaraarttym gngaracnmg ncay 24 107 25 DNA ArtificialSequence PCR primer O5436 107 ggaggaacac ggataagtgg gtaag 25 108 23 DNAArtificial Sequence APCR primer O5437 108 aggatttggc tggggacttt tcc 23109 35 DNA Artificial Sequence PCR primer O18016 109 acgcgtcgacccatcatcac cgagatcgac tcgac 35 110 37 DNA Artificial Sequence PCR primerO18017 110 ataagaatgc ggccgctcat tgttcgctca ctgggag 37 111 20 DNAArtificial Sequence PCR primer O18035 111 gctgacagaa cgggtgccga 20 11220 DNA Artificial Sequence PCR primer O18036 112 tgaccgtttc tggataggca20 113 24 DNA Artificial Sequence PCR primer O18065 113 ggccggcggggcgaaccaag ttcc 24 114 24 DNA Artificial Sequence PCR primer O18066 114aggcagagtc ggcgaagttg aggc 24 115 670 DNA Secale cereale Rye CBF20 gene115 cgtcgaccca cgcgtccgga tccatcgatc aaacctctca acacagctgc tgattcttcc 60agcactccac acttacaagc agcctcgatc tccgctagct ctagacctag atgccgtctg 120gtcaggaggg gcaacggcac aggacggtga ggtcggagcc gccgggcggt gggtctgcga 180ggtgcgcgtg ctcgggatga ggggctccag gctctggctt ggcaccttcg tcaccgcgga 240gatggcggcg cgcgcccacg acgccgccgt gctcgcgctc tccggccgca aggcctgcct 300caacttcgcc gactccgcct ggcggatgct gcccgtgctc gcggctggct ccttcggctt 360cggcagcgcg cgggagatca agaccgccgt cgccgtcgcc gtcctcgcgt tccagcggca 420gcagatcatt cttccagtag cccgcccggc ggaggagccg gccgacgtcc cgagcggcgc 480gctgttctcc atgtcatcag gcgacttgct ggagctcgac gaggagcagt ggtttggcgg 540catggttgcc gggtcctact acgagagctt ggcgcagggg atgctcgtcg agccgccgga 600cgccggagcg tggcgagagg acagcgagca cagcggcgtg gcggagacgc agacgccgtt 660gtggagctaa 670 116 222 PRT Secale cereale Rye CBF20 polypeptide 116 ValAsp Pro Arg Val Arg Ile His Arg Ser Asn Leu Ser Thr Gln Leu 1 5 10 15Leu Ile Leu Pro Ala Leu His Thr Tyr Lys Gln Pro Arg Ser Pro Leu 20 25 30Ala Leu Asp Leu Asp Ala Val Trp Ser Gly Gly Ala Thr Ala Gln Asp 35 40 45Gly Glu Val Gly Ala Ala Gly Arg Trp Val Cys Glu Val Arg Val Leu 50 55 60Gly Met Arg Gly Ser Arg Leu Trp Leu Gly Thr Phe Val Thr Ala Glu 65 70 7580 Met Ala Ala Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser Gly Arg 85 9095 Lys Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu Pro Val 100105 110 Leu Ala Ala Gly Ser Phe Gly Phe Gly Ser Ala Arg Glu Ile Lys Thr115 120 125 Ala Val Ala Val Ala Val Leu Ala Phe Gln Arg Gln Gln Ile IleLeu 130 135 140 Pro Val Ala Arg Pro Ala Glu Glu Pro Ala Asp Val Pro SerGly Ala 145 150 155 160 Leu Phe Ser Met Ser Ser Gly Asp Leu Leu Glu LeuAsp Glu Glu Gln 165 170 175 Trp Phe Gly Gly Met Val Ala Gly Ser Tyr TyrGlu Ser Leu Ala Gln 180 185 190 Gly Met Leu Val Glu Pro Pro Asp Ala GlyAla Trp Arg Glu Asp Ser 195 200 205 Glu His Ser Gly Val Ala Glu Thr GlnThr Pro Leu Trp Ser 210 215 220 117 813 DNA Secale cereale Rye CBF28gene 117 atggacgtcg ccgacatcgc ctccccgtct ggccagcagg agcaggggcaccggacggtg 60 tcgtcggagc cgccgaagcg ccccgcgggg cggaccaagt tccacgagacgcgccacccg 120 ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagt gggtgtgcgaggtgcgcgtg 180 cccgggatca agggctccag gctctggctc ggcaccttca acacggccgagatggcggcg 240 cgcgcccacg acgcagccgt gctcgcgctc tcctgccgcg ccgcctgcctcaacttcgcc 300 gactccgcct ggcggatgct gcccgtgctc gcggccgggt cgttcgggttcggcagcccg 360 cgggagatca aggcagccgt cgccgtcgcc gtcatcgcgt tccagcggaagcagattatt 420 ccggtcgccg tcgccgtcgt ggcgctccag cagcagcagg ttccagtcgccgtcgccgtc 480 gttgcgctaa agcagaagca ggttccggtc gctgtggccg tcgtggcgctccagcagctg 540 catgttccgg tagccgtcgc cgtcgtggcg ctccagcagc agcagattattcttccagtc 600 gcgtgcctgg cgcccgagtt ttacatgtct tccggcgacc tgctggagctcgacgaggag 660 cactggtttg gcggcatgga cgccgggtcg tactacgcga gcttggcgcaggggatgctc 720 gtggctccgc cggacgaaag agcgaggccg gagaacggcg agcaagagcggcgtccagac 780 gccgctatgg agctgtttgt tcgactaatt tag 813 118 270 PRTSecale cereale Rye CBF28 polypeptide 118 Met Asp Val Ala Asp Ile Ala SerPro Ser Gly Gln Gln Glu Gln Gly 1 5 10 15 His Arg Thr Val Ser Ser GluPro Pro Lys Arg Pro Ala Gly Arg Thr 20 25 30 Lys Phe His Glu Thr Arg HisPro Leu Tyr Arg Gly Val Arg Arg Arg 35 40 45 Gly Arg Val Gly Gln Trp ValCys Glu Val Arg Val Pro Gly Ile Lys 50 55 60 Gly Ser Arg Leu Trp Leu GlyThr Phe Asn Thr Ala Glu Met Ala Ala 65 70 75 80 Arg Ala His Asp Ala AlaVal Leu Ala Leu Ser Cys Arg Ala Ala Cys 85 90 95 Leu Asn Phe Ala Asp SerAla Trp Arg Met Leu Pro Val Leu Ala Ala 100 105 110 Gly Ser Phe Gly PheGly Ser Pro Arg Glu Ile Lys Ala Ala Val Ala 115 120 125 Val Ala Val IleAla Phe Gln Arg Lys Gln Ile Ile Pro Val Ala Val 130 135 140 Ala Val ValAla Leu Gln Gln Gln Gln Val Pro Val Ala Val Ala Val 145 150 155 160 ValAla Leu Lys Gln Lys Gln Val Pro Val Ala Val Ala Val Val Ala 165 170 175Leu Gln Gln Leu His Val Pro Val Ala Val Ala Val Val Ala Leu Gln 180 185190 Gln Gln Gln Ile Ile Leu Pro Val Ala Cys Leu Ala Pro Glu Phe Tyr 195200 205 Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu His Trp Phe Gly210 215 220 Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly MetLeu 225 230 235 240 Val Ala Pro Pro Asp Glu Arg Ala Arg Pro Glu Asn GlyGlu Gln Glu 245 250 255 Arg Arg Pro Asp Ala Ala Met Glu Leu Phe Val ArgLeu Ile 260 265 270 119 807 DNA Secale cereale Rye CBF46 gene 119atggacgtcg ccgacatcgc ctcccggtct ggccagcagc agcaggggca ccggaccgtg 60tcgtcggagc cgccgaagcg ccccgcgggg aggaccaagt tccacgagac gcgccacccg 120ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagt gggtgtgcga ggtgcgcgtt 180cccgggatca agggctccag gctctggctc ggcaccttca acacggccga gatggcggcg 240cgcgcgcacg acgccgccgt gctcgcgctc tccggccgca aagcctgcct caacttcgcc 300gactccgcct ggcggatgct gcccgtgctc gcggccggct ccttcggctt tgatagcgcg 360cgggaggtca aggccgccgt cgccgtcgcc gtcgtcgcgt tccagcggaa acagattatt 420ccagtcgccg tcgctgtcgt tgctctccag aagcagcagg ttccggtcgc cgtggccatc 480gtggcgctcc agcagaggca ggttccggtc gccgtcgccg tcgtggcgct ccagaagctg 540caggttccgg tcgccgtcgc cgtcgtagcg ctccagaaga agcagattat tcttccagcc 600gcgtgcctgg cgccggagtt ttacatgtct tccggcgacc tgttggagct cgacgaggag 660cagtggtttg gcggcatgga cgccgggtcg tactacgcca gcttggcgca ggggatgctc 720gtggcgccgc cggacgacag agcgaggccg gagaacggcg agcagagcgg cgtccagact 780ccgctatgga gctgcttgtt cgactaa 807 120 268 PRT Secale cereale Rye CBF46polypeptide 120 Met Asp Val Ala Asp Ile Ala Ser Arg Ser Gly Gln Gln GlnGln Gly 1 5 10 15 His Arg Thr Val Ser Ser Glu Pro Pro Lys Arg Pro AlaGly Arg Thr 20 25 30 Lys Phe His Glu Thr Arg His Pro Leu Tyr Arg Gly ValArg Arg Arg 35 40 45 Gly Arg Val Gly Gln Trp Val Cys Glu Val Arg Val ProGly Ile Lys 50 55 60 Gly Ser Arg Leu Trp Leu Gly Thr Phe Asn Thr Ala GluMet Ala Ala 65 70 75 80 Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser GlyArg Lys Ala Cys 85 90 95 Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu ProVal Leu Ala Ala 100 105 110 Gly Ser Phe Gly Phe Asp Ser Ala Arg Glu ValLys Ala Ala Val Ala 115 120 125 Val Ala Val Val Ala Phe Gln Arg Lys GlnIle Ile Pro Val Ala Val 130 135 140 Ala Val Val Ala Leu Gln Lys Gln GlnVal Pro Val Ala Val Ala Ile 145 150 155 160 Val Ala Leu Gln Gln Arg GlnVal Pro Val Ala Val Ala Val Val Ala 165 170 175 Leu Gln Lys Leu Gln ValPro Val Ala Val Ala Val Val Ala Leu Gln 180 185 190 Lys Lys Gln Ile IleLeu Pro Ala Ala Cys Leu Ala Pro Glu Phe Tyr 195 200 205 Met Ser Ser GlyAsp Leu Leu Glu Leu Asp Glu Glu Gln Trp Phe Gly 210 215 220 Gly Met AspAla Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met Leu 225 230 235 240 ValAla Pro Pro Asp Asp Arg Ala Arg Pro Glu Asn Gly Glu Gln Ser 245 250 255Gly Val Gln Thr Pro Leu Trp Ser Cys Leu Phe Asp 260 265 121 639 DNASecale cereale Rye CBF7 gene 121 atggacgccg ccgacgccgg ctccccccgttttgggcaca ggacggtgtg ctcggagccg 60 cccaagaggc cggcagggcg gaccaagtttaaggagaccc gccacccgct gtaccgcggc 120 gtgcggcggc ggggtcggct cgggcagtgggtgtgcgagg tgcgcgtgcg cggcgcgcaa 180 gggtacaggc tctggctcgg cacattcaccaccgccgaga tggcggcgcg cgcgcacgac 240 tccgccgtgc tcgcgctcct cgaccgcgccgcttgcctca acttcgccga ctccgcctgg 300 cggatgctgc ccgtcctcgc ggcaggctcgtcccgcttca gcagcgcgcg ggaaatcaag 360 gacgccgtcg ccgtcgccgt cgtggagttccagcggcagc gccccttcgt gtccacgtcg 420 gagacggccg acggcgagaa ggacgtccaaggctcgccga ggccgagcga gctgtccacg 480 tccagcgact tgttggacga gcactggtttagcggcatgg acgccggctc ttactacgcg 540 agcttggcgc aggggatgct catggagccgccggccgcca gagcgtggag cgaggatggc 600 ggcgaataca gcggcgtcca cacgccgctttggaactag 639 122 212 PRT Secale cereale Rye CBF7 polypeptide 122 MetAsp Ala Ala Asp Ala Gly Ser Pro Arg Phe Gly His Arg Thr Val 1 5 10 15Cys Ser Glu Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Lys Glu 20 25 30Thr Arg His Pro Leu Tyr Arg Gly Val Arg Arg Arg Gly Arg Leu Gly 35 40 45Gln Trp Val Cys Glu Val Arg Val Arg Gly Ala Gln Gly Tyr Arg Leu 50 55 60Trp Leu Gly Thr Phe Thr Thr Ala Glu Met Ala Ala Arg Ala His Asp 65 70 7580 Ser Ala Val Leu Ala Leu Leu Asp Arg Ala Ala Cys Leu Asn Phe Ala 85 9095 Asp Ser Ala Trp Arg Met Leu Pro Val Leu Ala Ala Gly Ser Ser Arg 100105 110 Phe Ser Ser Ala Arg Glu Ile Lys Asp Ala Val Ala Val Ala Val Val115 120 125 Glu Phe Gln Arg Gln Arg Pro Phe Val Ser Thr Ser Glu Thr AlaAsp 130 135 140 Gly Glu Lys Asp Val Gln Gly Ser Pro Arg Pro Ser Glu LeuSer Thr 145 150 155 160 Ser Ser Asp Leu Leu Asp Glu His Trp Phe Ser GlyMet Asp Ala Gly 165 170 175 Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met LeuMet Glu Pro Pro Ala 180 185 190 Ala Arg Ala Trp Ser Glu Asp Gly Gly GluTyr Ser Gly Val His Thr 195 200 205 Pro Leu Trp Asn 210 123 807 DNASecale cereale Rye CBF71 123 atggacgtcg ccgacatcgc ctcccggtct ggccagcagcagcaggggca ccggaccgtg 60 tcgtcggagc cgccgaagcg ccccgcgggg aggaccaagttccacgagac gcgccacccg 120 ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagtgggtgtgcga ggtgcgcgtg 180 cccgggatca agggctccag gctctggctc ggcaccttcaacacggccga gatggcggcg 240 cgcgcgcacg acgctgccgt gctcgcgctc tccggccgcgccgcctgcct caacttcgcc 300 gactccgcct ggcggatgct gcccgtgctc gcggccggctccttcggctt tgatagcgcg 360 cgggaggtca aggccgccgt cgccgtcgcc gtcgtcgcgttccagcggaa acagattatt 420 ccagtcgccg tcgctgtcgt tgctctccag aagcagcaggttccggtcgc cgtggccgtc 480 gtggcgctcc agcagaggca ggttccggtc accgtcgccgtcgtggcgct ccagaagctg 540 caggttccgg tcgccgtcgc cgtcgtggcg ctccagaagaagcagattat tcttccagcc 600 gcgtgtctgg cgccggagtt ttacatgtct tccggcgacctgttggagct cgacgaggag 660 cagtggtttg gcggcatgga cgccgggtcg tactacgccagcttggcgca ggggatgctc 720 gtggcgccgc cggacgacag agcgaggccg gagaacggcgagcagagcgg cgtccagact 780 ccgctatgga gctgcttgtt cgactaa 807 124 268 PRTSecale cereale Rye CBF71 polypeptide 124 Met Asp Val Ala Asp Ile Ala SerArg Ser Gly Gln Gln Gln Gln Gly 1 5 10 15 His Arg Thr Val Ser Ser GluPro Pro Lys Arg Pro Ala Gly Arg Thr 20 25 30 Lys Phe His Glu Thr Arg HisPro Leu Tyr Arg Gly Val Arg Arg Arg 35 40 45 Gly Arg Val Gly Gln Trp ValCys Glu Val Arg Val Pro Gly Ile Lys 50 55 60 Gly Ser Arg Leu Trp Leu GlyThr Phe Asn Thr Ala Glu Met Ala Ala 65 70 75 80 Arg Ala His Asp Ala AlaVal Leu Ala Leu Ser Gly Arg Ala Ala Cys 85 90 95 Leu Asn Phe Ala Asp SerAla Trp Arg Met Leu Pro Val Leu Ala Ala 100 105 110 Gly Ser Phe Gly PheAsp Ser Ala Arg Glu Val Lys Ala Ala Val Ala 115 120 125 Val Ala Val ValAla Phe Gln Arg Lys Gln Ile Ile Pro Val Ala Val 130 135 140 Ala Val ValAla Leu Gln Lys Gln Gln Val Pro Val Ala Val Ala Val 145 150 155 160 ValAla Leu Gln Gln Arg Gln Val Pro Val Thr Val Ala Val Val Ala 165 170 175Leu Gln Lys Leu Gln Val Pro Val Ala Val Ala Val Val Ala Leu Gln 180 185190 Lys Lys Gln Ile Ile Leu Pro Ala Ala Cys Leu Ala Pro Glu Phe Tyr 195200 205 Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu Gln Trp Phe Gly210 215 220 Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly MetLeu 225 230 235 240 Val Ala Pro Pro Asp Asp Arg Ala Arg Pro Glu Asn GlyGlu Gln Ser 245 250 255 Gly Val Gln Thr Pro Leu Trp Ser Cys Leu Phe Asp260 265 125 240 DNA Triticum aestivum Wheat CBF gene 125 agtgattggccggcggggcg aaccaagttc cgtgagacac gacacccgct gtaccgcggc 60 gtgcggcgccgtggccgggt cgggcagtgg gtgtgcgagg tgcgcgtgcc aggagtgaag 120 ggctccaggctctggctcgg caccttcacc accgccgaga tggcggcgcg cgcgcacgac 180 gccgcggtgctcgcgctctc cggccgcgcc gcctgcctca acttcgccga ctctgcctaa 240 126 76 PRTTriticum aestivum Wheat CBF polypeptide 126 Pro Ala Gly Arg Thr Lys PheArg Glu Thr Arg His Pro Leu Tyr Arg 1 5 10 15 Gly Val Arg Arg Arg GlyArg Val Gly Gln Trp Val Cys Glu Val Arg 20 25 30 Val Pro Gly Val Lys GlySer Arg Leu Trp Leu Gly Thr Phe Thr Thr 35 40 45 Ala Glu Met Ala Ala ArgAla His Asp Ala Ala Val Leu Ala Leu Ser 50 55 60 Gly Arg Ala Ala Cys LeuAsn Phe Ala Asp Ser Ala 65 70 75 127 705 DNA Glycine max Soy CBF gene127 atgtttacct tgaatcattc ttctgatttg taccatgttt cccctgagct ctcatcttcc 60ttggacacat cctcgccggc ttcggagggc tctcgtggcg tggcattttc cgacgaggag 120gtgcggctgg cggtgaggca cccgaagaag cgggcaggtc ggaagaagtt ccgggagacg 180cgccacccgg tgtaccgggg ggtgaggagg aggaactcgg ataagtgggt gtgtgaggtg 240agggagccca acaagaagac caggatttgg ctggggactt tccccacgcc ggagatggcg 300gctcgggcgc acgacgtggc ggcaatggcc ctgaggggcc ggtatgcctg tctaaacttt 360gctgactcgg cctggcggtt acctgttccc gccacggccg aggcaaagga tatacagaag 420gcagcagcag aagctgccca ggctttcaga ccagatcaaa ccttaaaaaa tgctaataca 480aggcaggagt gtgtggaggc ggtggcggtg gcggtggcgg agacaacaac ggcgacggca 540caaggggtgt tttatatgga ggaagaagag caggtgttgg atatgcctga gttgcttagg 600aatatggtgc tcatgtcccc aacacattgc ttagggtatg agtatgaaga tgctgacttg 660gatgcccaag atgctgaggt gtcactatgg aatttctcaa tttaa 705 128 234 PRTGlycine max Soy CBF polypeptide 128 Met Phe Thr Leu Asn His Ser Ser AspLeu Tyr His Val Ser Pro Glu 1 5 10 15 Leu Ser Ser Ser Leu Asp Thr SerSer Pro Ala Ser Glu Gly Ser Arg 20 25 30 Gly Val Ala Phe Ser Asp Glu GluVal Arg Leu Ala Val Arg His Pro 35 40 45 Lys Lys Arg Ala Gly Arg Lys LysPhe Arg Glu Thr Arg His Pro Val 50 55 60 Tyr Arg Gly Val Arg Arg Arg AsnSer Asp Lys Trp Val Cys Glu Val 65 70 75 80 Arg Glu Pro Asn Lys Lys ThrArg Ile Trp Leu Gly Thr Phe Pro Thr 85 90 95 Pro Glu Met Ala Ala Arg AlaHis Asp Val Ala Ala Met Ala Leu Arg 100 105 110 Gly Arg Tyr Ala Cys LeuAsn Phe Ala Asp Ser Ala Trp Arg Leu Pro 115 120 125 Val Pro Ala Thr AlaGlu Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 130 135 140 Ala Ala Gln AlaPhe Arg Pro Asp Gln Thr Leu Lys Asn Ala Asn Thr 145 150 155 160 Arg GlnGlu Cys Val Glu Ala Val Ala Val Ala Val Ala Glu Thr Thr 165 170 175 ThrAla Thr Ala Gln Gly Val Phe Tyr Met Glu Glu Glu Glu Gln Val 180 185 190Leu Asp Met Pro Glu Leu Leu Arg Asn Met Val Leu Met Ser Pro Thr 195 200205 His Cys Leu Gly Tyr Glu Tyr Glu Asp Ala Asp Leu Asp Ala Gln Asp 210215 220 Ala Glu Val Ser Leu Trp Asn Phe Ser Ile 225 230 129 2396 DNAArabidopsis thaliana Artificial fusion between rab18 promoter and CBF1gene 129 aagcttcaaa ttctgaatat tcacatatca aaaaagtgat gcgagggagagaagggtatt 60 tcatcttact tgcagcgata gaactccttg tggtagaagt gacatataactttttcactc 120 cttgtgactt ctcccaagaa gtcgccttcg ctaacttctc ggtattcaccatgtccttgt 180 cttttgaatg cttctctctt ttccacttct ctctgtccag acacatcttgggattagatc 240 agtccatact aatgaagaca aaagtgttaa ccaaatatag ataatgagagaatgtggggg 300 gtgaagttga accctgagtg ctgcaatcct atctgcgtgc aacttttctagctctggatc 360 ctacagaaca agagcaagtc agtctcccat agagacagaa gagaaacataacatttcatt 420 gtacagatta taaaagaaca attcaaagag agcccccatt gaacttacatccatcaattc 480 gtcaagatca acttcctcgt tgacaggtct tgatccttgt gccttttcatttgcaagaac 540 ttcctgcaca acaaaaatac ggaaatgttt ttgatccaag aacatcaaactctaattgca 600 attgtaacca acaaaaaaac acttaaaatt cgctatacac tatcaaatttcctgacacgt 660 agacctcatg tcagcacaaa tagaatctag cctatctctg ttaattgggttaccaaacaa 720 catgagattg attatgtgga aaaccaagca cattatttac cctttgaaaatacgcgaaat 780 caagatccaa gaagaattta tggagaacag cttcgaagta cgaaataaacaatgaagatt 840 aaattacctt tttataatct ctagcagctg ccgccaatac attcccgaatgccagattcg 900 agagggtcga cttcaccgta tccggatcca tctctttacc aaccaactaatccaactcag 960 aaaattttaa aatctcaatc aaaaatccct ctaagatagc cagagaagagattgtaaaca 1020 aggatttgaa atctggtgca gagaggagaa actccccgac aatgaacaccaacgatctaa 1080 acgcggcgtt tggtaaaagt tgagtaaatt ttgttagggc ttagttttagtccatgggct 1140 aattagtaag tgatttacgg cccacacatg agcccaaatg tttcagacccagccaagttt 1200 cttcaaattc acccaatcaa cgacgatgta cgtgtgtatg aaaatcattaacacgacgca 1260 tcgctttcga ggaggagcat tacgtgtcct gttagctacg ataatgttagtaccgccaca 1320 aagaaaagga tagatatttt gctttccagc accctgtcat gggattgatatgaacacgta 1380 cttggtatcg acatgaaagc tcaaaaataa attcaatccg attcctttagtgatatcaga 1440 agttcatttt aaatacgaac acgtatggcg aaacaccacg ccgacattttctgctgctgc 1500 cacgcgtcac tttccaaata ttgattcatt aaactaatag ttgatccatatccgaaaccg 1560 gactataaaa ctatcttcaa tgcgttaacg aatcttcatc gatcaaactcatcaaagtct 1620 aatatcacaa agaaagagtt tttttaacta gcttagctca aagtgtttgcttaagacaag 1680 aagaacgaat tcaatgaact cattttcagc tttttctgaa atgtttggctccgattacga 1740 gcctcaaggc ggagattatt gtccgacgtt ggccacgagt tgtccgaagaaaccggcggg 1800 ccgtaagaag tttcgtgaga ctcgtcaccc aatttacaga ggagttcgtcaaagaaactc 1860 cggtaagtgg gtttctgaag tgagagagcc aaacaagaaa accaggatttggctcgggac 1920 tttccaaacc gctgagatgg cagctcgtgc tcacgacgtc gctgcattagccctccgtgg 1980 ccgatcagca tgtctcaact tcgctgactc ggcttggcgg ctacgaatcccggagtcaac 2040 atgcgccaag gatatccaaa aagcggctgc tgaagcggcg ttggcttttcaagatgagac 2100 gtgtgatacg acgaccacgg atcatggcct ggacatggag gagacgatggtggaagctat 2160 ttatacaccg gaacagagcg aaggtgcgtt ttatatggat gaggagacaatgtttgggat 2220 gccgactttg ttggataata tggctgaagg catgctttta ccgccgccgtctgttcaatg 2280 gaatcataat tatgacggcg aaggagatgg tgacgtgtcg ctttggagttactaatattc 2340 gatagtcgtt tccatttttg tactatagtt tgaaaatatt ctagttccgcggccgc 2396 130 2655 DNA Arabidopsis thaliana Artificial fusion betweenDREB2a promoter and CBF1 gene 130 aagcttgtaa tcgataacct aaatcattgtaatgaatgcg ttcctcccta tcgatcctag 60 gccttagaca atgctgaatg attcatagccacgcgaataa cctattcata ctaatatgga 120 aagaaagaag ccaaacttac agagctcttctcacggtcgt cgaagaaaag agagtctaca 180 gtcagcaaca aaattagtgt tgccatcatggcatcatctt gcagcttttt ccgcacaaat 240 aatttatcat ccaaatgttg ttcactaaaccaaaaacaaa caagcattac agcgaagaaa 300 ccaactcgta gatacagatt tccaaattcatgccttattt agaccaataa aaactgaaat 360 ttctcttcag cgaaaaaaaa aacaaacaaactgaaatttc atatagatcc agaagataga 420 aacttgtagg ctcaatcgac tagactagaagatgctcacc cgatcgtgct tgagatagcg 480 agatagtagc aacaccgacg gtagaaataaaatggacgac acccatccaa tgggctaatt 540 tagattaacg ggctttaagg gtttgataatggatgttaat taactgaggc acatgggatt 600 gtatcacgta ggcaatgggt ttgataatggatgttaagta actaaggccc atgaggttga 660 agtacgtagg caattgcgtg agcttacgttagcgatgccg ttagagacac gtagtggatg 720 aagtggcttt ggttagcaaa ggacacatgaggcacatgca aaggctataa atgactgctg 780 ctttgctaca acttgcgatt cccaaattttataaggtaat ggaccctatc acctctgctc 840 gaagctaagc cacccaagtt tgagcttcaccatttgacac gtctctagct aatacttaat 900 gcttaaactt taaactaata cttcatgtttaagacatttc tggctgacac atttatgaat 960 tcgctctatg tcgtacgtac accggaaacctttaatttta caatattaac cgtgatcttt 1020 ttaaaaatat ctatataaac gataagtatctcatcaaaac aaaattgaat aatgtgcacg 1080 tttgtaaaaa ctagaaaaac aggcaataaacatcatcatc caattacaca tctagtaagt 1140 gtgatgcagt ggcaaactgg caataaaccaagaaaaatcg agaaagagca gatgagacag 1200 tgttgtgttg tcagggttaa taaaaaaaaaatgaagatat tttaaaattt cataatattt 1260 aaataatgaa gtagttttat gatcttatccataaatcaat tttaaaaagg tttaaacttt 1320 acttttccgt atcaacagcg tgtttcgagaagattcggga ggacactcgt cgaacggaaa 1380 agtcgtctaa gcctttatgt tcgaatcaaaaactgacacg taaccttgct ctcaaacaga 1440 aaaataaaat aatgttagaa aaatctagagaaggctataa atactccgta gatactttgt 1500 cttccttaga tattttgatt tctgctaaagctgtctgata aaaagaagag gaaaactcga 1560 aaaagctaca cacaagaaga agaagaaaagatacgagcaa gaagactaaa cacgaaagcg 1620 atttatcaac tcgaaggaag agactttgattttcaaattt cgtcccctat aggtacgttg 1680 atttctgatt tcttacaaat tttcaattaatttctctgat taggtctcgt caattggtga 1740 ttctagggtt tatcttcgtc tttaggttttcgattatttc cttttaggta tagctgtgaa 1800 attgggaaat tttaggtgtt cctaatttgataacaaataa gtaaatcgat ctgattttga 1860 accaagttta agttctcttg ccttatgtttcagcttgtgt tctgatattg aagtgttgtt 1920 gtattgtaga ttgtgttgtt tctgggaaggccatggactc attttcagct ttttctgaaa 1980 tgtttggctc cgattacgag cctcaaggcggagattattg tccgacgttg gccacgagtt 2040 gtccgaagaa accggcgggc cgtaagaagtttcgtgagac tcgtcaccca atttacagag 2100 gagttcgtca aagaaactcc ggtaagtgggtttctgaagt gagagagcca aacaagaaaa 2160 ccaggatttg gctcgggact ttccaaaccgctgagatggc agctcgtgct cacgacgtcg 2220 ctgcattagc cctccgtggc cgatcagcatgtctcaactt cgctgactcg gcttggcggc 2280 tacgaatccc ggagtcaaca tgcgccaaggatatccaaaa agcggctgct gaagcggcgt 2340 tggcttttca agatgagacg tgtgatacgacgaccacgga tcatggcctg gacatggagg 2400 agacgatggt ggaagctatt tatacaccggaacagagcga aggtgcgttt tatatggatg 2460 aggagacaat gtttgggatg ccgactttgttggataatat ggctgaaggc atgcttttac 2520 cgccgccgtc tgttcaatgg aatcataattatgacggcga aggagatggt gacgtgtcgc 2580 tttggagtta ctaatattcg atagtcgtttccatttttgt actatagttt gaaaatattc 2640 tagttccgcg gccgc 2655 131 21 DNAArtificial sequence O6792 Synthetic oligonucleotide primer 131gcttgtccat tatcctagaa t 21 132 28 DNA Artificial sequence O6793Synthetic oligonucleotide primer 132 cggaattcgt tcttcttgtc ttaagcaa 28133 35 DNA Artificial sequence O6805 Synthetic oligonucleotide primer133 cggaattcaa tgaactcatt ttcagctttt tctga 35 134 43 DNA Artificialsequence O6806 Synthetic oligonucleotide primer 134 ataagaatgcggccgcggaa ctagaatatt ttcaaactat agt 43 135 32 DNA Artificial sequenceO6798 Synthetic oligonucleotide primer 135 gcccaagctt gtaatcgataacctaaatca tt 32 136 31 DNA Artificial sequence O6799 Syntheticoligonucleotide primer 136 aactgccatg gccttcccag aaacaacaca a 31 137 62PRT Arabidopsis thaliana 137 His Pro Ile Tyr Arg Gly Val Arg Gln Arg AsnSer Gly Lys Trp Val 1 5 10 15 Cys Glu Leu Arg Glu Pro Asn Lys Lys ThrArg Ile Trp Leu Gly Thr 20 25 30 Phe Gln Thr Ala Glu Met Ala Ala Arg AlaHis Asp Val Ala Ala Ile 35 40 45 Ala Leu Arg Gly Arg Ser Ala Cys Leu AsnPhe Ala Asp Ser 50 55 60 138 62 PRT Arabidopsis thaliana 138 His Pro IleTyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val 1 5 10 15 Cys GluVal Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 20 25 30 Phe GlnThr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala LeuArg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 139 62 PRTArabidopsis thaliana 139 His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn SerGly Lys Trp Val 1 5 10 15 Ser Glu Val Arg Glu Pro Asn Lys Lys Thr ArgIle Trp Leu Gly Thr 20 25 30 Phe Gln Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn PheAla Asp Ser 50 55 60 140 62 PRT Brassica juncea 140 His Pro Ile Tyr ArgGly Val Arg Gln Arg Asn Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Pro Thr ValGlu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg GlyArg Ser Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 141 62 PRT Brassicaoleracea 141 His Pro Val Tyr Arg Gly Val Arg Leu Arg Asn Ser Gly Lys TrpVal 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp LeuGly Thr 20 25 30 Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val AlaAla Ile 35 40 45 Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser50 55 60 142 62 PRT Raphanus sativus 142 His Pro Ile Tyr Arg Gly Val ArgLeu Arg Asn Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro AsnLys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Leu Thr Ala Glu Ile AlaAla Arg Ala His Asp Val Ala Ala Ile 35 40 45 Ala Leu Arg Gly Lys Ser AlaCys Leu Asn Phe Ala Asp Ser 50 55 60 143 62 PRT Brassica juncea 143 HisPro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15Cys Glu Val Arg Glu Pro Asn Lys Arg Ser Arg Ile Trp Leu Gly Thr 20 25 30Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 35 40 45Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 144 62PRT Brassica napus 144 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser ArgIle Trp Leu Gly Thr 20 25 30 Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala HisAsp Val Ala Ala Ile 35 40 45 Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn PheAla Asp Ser 50 55 60 145 62 PRT Brassica juncea 145 His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly Thr 20 25 30 Phe Leu Thr AlaGlu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 35 40 45 Ala Leu Arg GlyLys Ser Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 146 62 PRT Brassicanapus 146 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys TrpVal 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp ProGly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val AlaAla Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser50 55 60 147 62 PRT Brassica napus 147 His Pro Ile Tyr Arg Gly Val ArgLeu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro AsnLys Lys Ser Arg Ile Trp Pro Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly AlaCys Leu Asn Tyr Ala Asp Ser 50 55 60 148 62 PRT Brassica napus 148 HisPro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser 50 55 60 149 62PRT Brassica napus 149 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser ArgIle Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn TyrAla Asp Ser 50 55 60 150 62 PRT Brassica napus 150 His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr AlaGlu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg GlyArg Gly Ala Cys Leu Asn Tyr Ala Asp Ser 50 55 60 151 62 PRT Brassicaoleracea 151 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys TrpVal 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp LeuGly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val AlaAla Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser50 55 60 152 62 PRT Brassica rapa 152 His Pro Ile Tyr Arg Gly Val ArgLeu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro AsnLys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly AlaCys Leu Asn Tyr Ala Asp Ser 50 55 60 153 62 PRT Brassica napus 153 HisPro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 154 62PRT Brassica napus 154 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser ArgIle Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala HisAsp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn PheAla Asp Ser 50 55 60 155 62 PRT Brassica oleracea 155 His Pro Ile TyrArg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu ValArg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys ThrAla Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu ArgGly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 156 62 PRT Brassicarapa 156 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu GlyThr 20 25 30 Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala AlaLeu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 5055 60 157 62 PRT Brassica rapa 157 His Pro Ile Tyr Arg Gly Val Arg LeuArg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn LysLys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Ile Ala AlaArg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala CysLeu Asn Phe Ala Asp Ser 50 55 60 158 62 PRT Brassica rapa 158 His ProIle Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 CysGlu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 PheLys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 AlaLeu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 159 62 PRTBrassica rapa 159 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser GlyLys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg IleTrp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His AspVal Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe AlaAsp Ser 50 55 60 160 62 PRT Brassica rapa 160 His Pro Ile Tyr Arg GlyVal Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg GluPro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala GluIle Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly ArgGly Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 161 62 PRT Brassica rapa161 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 510 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 2025 30 Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 3540 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60162 62 PRT Raphanus sativus 162 His Pro Ile Tyr Arg Gly Val Arg Leu ArgLys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys LysSer Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Ile Ala Ala ArgAla His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys LeuAsn Phe Ala Asp Ser 50 55 60 163 62 PRT Brassica oleracea 163 His ProIle Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 CysGlu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 PheLys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 AlaLeu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60 164 62 PRTBrassica oleracea 164 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys SerGly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Leu Asn Lys Lys Ser ArgIle Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn TyrAla Asp Ser 50 55 60 165 62 PRT Brassica napus 165 His Pro Ile Tyr ArgGly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly Thr 20 25 30 Phe Lys Thr AlaGlu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg GlyArg Gly Ala Arg Leu Asn Tyr Ala Asp Ser 50 55 60 166 63 PRT Zea mays 166His Pro Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly Arg Trp 1 5 1015 Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 20 2530 Thr Phe Ala Thr Pro Glu Ala Ala Ala Arg Ala His Asp Val Ala Ala 35 4045 Leu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser 50 55 60167 62 PRT Glycine max 167 His Pro Ile Tyr Ser Gly Val Arg Arg Arg AsnThr Asp Lys Trp Val 1 5 10 15 Ser Glu Val Arg Glu Pro Asn Lys Lys ThrArg Ile Trp Leu Gly Thr 20 25 30 Phe Pro Thr Pro Glu Met Ala Ala Arg AlaHis Asp Val Ala Ala Met 35 40 45 Ala Leu Arg Gly Arg Tyr Ala Cys Leu AsnPhe Ala Asp Ser 50 55 60 168 63 PRT Nicotiana tabacum 168 Gly Arg HisTyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Phe Ala 1 5 10 15 Ala GluIle Arg Asp Pro Ala Lys Asn Gly Ala Arg Val Trp Leu Gly 20 25 30 Thr TyrGlu Thr Ala Glu Glu Ala Ala Leu Ala Tyr Asp Lys Ala Ala 35 40 45 Tyr ArgMet Arg Gly Ser Lys Ala Leu Leu Asn Phe Pro His Arg 50 55 60 169 62 PRTArabidopsis thaliana 169 Arg Cys Ser Phe Arg Gly Val Arg Gln Arg Ile TrpGly Lys Trp Val 1 5 10 15 Ala Glu Ile Arg Glu Pro Asn Arg Gly Ser ArgLeu Trp Leu Gly Thr 20 25 30 Phe Pro Thr Ala Gln Glu Ala Ala Ser Ala TyrAsp Glu Ala Ala Lys 35 40 45 Ala Met Tyr Gly Pro Leu Ala Arg Leu Asn PhePro Arg Ser 50 55 60 170 62 PRT Arabidopsis thaliana 170 His Cys Ser PheArg Gly Val Arg Gln Arg Ile Trp Gly Lys Trp Val 1 5 10 15 Ala Glu IleArg Glu Pro Lys Ile Gly Thr Arg Leu Trp Leu Gly Thr 20 25 30 Phe Pro ThrAla Glu Lys Ala Ala Ser Ala Tyr Asp Glu Ala Ala Thr 35 40 45 Ala Met TyrGly Ser Leu Ala Arg Leu Asn Phe Pro Gln Ser 50 55 60 171 62 PRTArabidopsis thaliana 171 His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn TrpGly Lys Trp Val 1 5 10 15 Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser ArgIle Trp Leu Gly Thr 20 25 30 Phe Pro Ser Pro Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Leu 35 40 45 Ser Ile Lys Gly Ala Ser Ala Ile Leu Asn PhePro Asp Leu 50 55 60 172 46 PRT Brassica rapa 172 Met Asn Ser Val SerThr Phe Ser Glu Leu Leu Cys Ser Glu Asn Glu 1 5 10 15 Ser Pro Val AsnThr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 20 25 30 Pro Lys Lys ProAla Gly Arg Lys Lys Phe Gln Glu Thr Arg 35 40 45 173 46 PRT Brassicarapa 173 Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Lys1 5 10 15 Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala SerCys 20 25 30 Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg 3540 45 174 46 PRT Brassica napus 174 Met Asn Ser Val Ser Thr Phe Ser GluLeu Leu Arg Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Asn Thr Glu Gly GlyAsp Tyr Ile Leu Ala Ala Ser Cys 20 25 30 Pro Lys Lys Pro Ala Gly Arg LysLys Phe Gln Glu Thr Arg 35 40 45 175 47 PRT Arabidopsis thaliana 175 MetAsn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys Leu Ala Thr Ser 20 25 30Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 45 17647 PRT Arabidopsis thaliana 176 Met Asn Ser Phe Ser Ala Phe Ser Glu MetPhe Gly Ser Asp Tyr Glu 1 5 10 15 Ser Ser Val Ser Ser Gly Gly Asp TyrIle Pro Thr Leu Ala Ser Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg LysLys Phe Arg Glu Thr Arg 35 40 45 177 44 PRT Arabidopsis thaliana 177 MetAsn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15Pro Gln Gly Gly Asp Tyr Cys Pro Thr Leu Ala Thr Ser Cys Pro Lys 20 25 30Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 178 49 PRTBrassica napus 178 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly SerGlu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr TyrPro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys LysPhe Gln Glu Thr 35 40 45 Arg 179 49 PRT Brassica napus 179 Met Asn ThrPhe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 1 5 10 15 Ser ProVal Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala SerCys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg 18049 PRT Brassica oleracea 180 Met Asn Thr Phe Pro Ala Ser Thr Glu Met ValGly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly AspTyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly ArgLys Lys Phe Gln Glu Thr 35 40 45 Arg 181 49 PRT Brassica napus 181 MetAsn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 1 5 10 15Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45Arg 182 49 PRT Brassica napus 182 Met Asn Thr Phe Pro Ala Ser Thr GluMet Val Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val Ala GlyGly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro AlaGly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg 183 49 PRT Brassica napus183 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 1 510 15 Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 2025 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 3540 45 Arg 184 49 PRT Brassica rapa 184 Met Asn Thr Phe Pro Ala Ser ThrGlu Met Val Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val AlaGly Gly Asp Tyr Tyr Pro Met Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys ProAla Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg 185 49 PRT Brassicaoleracea 185 Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Ser Ser Glu AsnGlu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro MetLeu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe GlnGlu Thr 35 40 45 Arg 186 47 PRT Brassica oleracea 186 Met Thr Ser PheSer Thr Phe Ser Glu Leu Leu Gly Ser Glu His Glu 1 5 10 15 Ser Pro ValThr Leu Gly Glu Glu Tyr Cys Pro Lys Leu Ala Ala Ser 20 25 30 Cys Pro LysLys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 45 187 47 PRTRaphanus sativus 187 Met Thr Thr Phe Ser Thr Phe Ser Glu Met Leu Gly SerGlu Tyr Glu 1 5 10 15 Ser Pro Val Thr Leu Gly Gly Glu Tyr Cys Pro LysLeu Ala Ala Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe ArgGlu Thr Arg 35 40 45 188 45 PRT Brassica napus 188 Met Asn Ser Val SerThr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val GlyGly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 20 25 30 Lys Lys Pro AlaGly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 45 189 45 PRT Brassica napus189 Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 1 510 15 Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 2025 30 Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 45 19045 PRT Brassica oleracea 190 Met Asn Ser Val Ser Thr Phe Ser Glu Leu LeuGly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Gly Gly Asp Tyr Cys Pro MetLeu Ala Ala Ser Cys Pro 20 25 30 Lys Lys Pro Ala Gly Arg Lys Lys Phe ArgGlu Thr Arg 35 40 45 191 45 PRT Brassica rapa 191 Met Asn Ser Val SerThr Leu Ser Glu Val Leu Gly Ser Gln Asn Glu 1 5 10 15 Ser Pro Val GlyGly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 20 25 30 Lys Lys Pro AlaGly Arg Lys Lys Phe Arg Glu Thr Arg 35 40 45 192 49 PRT Brassica rapa192 Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 1 510 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 2025 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 3540 45 Arg 193 49 PRT Brassica rapa 193 Met Asp Ser Ile Ser Thr Phe ProGlu Leu Leu Gly Ser Glu Asn Glu 1 5 10 15 Ser Pro Val Thr Thr Val ValGly Gly Asp Tyr Cys Pro Arg Leu Ala 20 25 30 Ala Ser Cys Pro Lys Lys ProAla Gly Arg Lys Lys Phe Gln Glu Thr 35 40 45 Arg 194 49 PRT Raphanussativus 194 Met Asp Ser Ile Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu AsnGlu 1 5 10 15 Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Phe Pro ArgLeu Ala 20 25 30 Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe GlnGlu Thr 35 40 45 Arg 195 50 PRT Brassica napus 195 Ala Trp Arg Leu ArgIle Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala AlaGlu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met GlnAsn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 35 40 45 Ser Gln 50 19650 PRT Brassica napus 196 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr CysHis Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala PheGlu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Gln Asn Gly Leu Asn Met GluGlu Thr Thr Ala Val Ala 35 40 45 Ser Gln 50 197 50 PRT Brassica rapa 197Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 1015 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 2530 Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala 35 4045 Ser Gln 50 198 50 PRT Brassica napus 198 Ala Trp Arg Leu Arg Ile ProGlu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu AlaAla Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Gln Asn GlyGln Asn Met Glu Glu Thr Thr Ala Val Ala 35 40 45 Ser Gln 50 199 44 PRTBrassica napus 199 Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His LysAsp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu AlaGlu Lys Ser Asp 20 25 30 Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln35 40 200 44 PRT Brassica napus 200 Ala Ser Arg Leu Arg Ile Pro Glu ThrThr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala LeuAla Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Glu Glu Thr Met AlaVal Ala Ser Gln 35 40 201 44 PRT Brassica oleracea 201 Ala Trp Arg LeuArg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala AlaAla Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr MetGlu Glu Thr Met Ala Val Ala Ser Gln 35 40 202 50 PRT Brassica napus 202Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 1015 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 2530 Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 35 4045 Ser Gln 50 203 50 PRT Brassica oleracea 203 Ala Trp Arg Leu Arg IlePro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala GluAla Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Ala Thr Met Gln AsnGly Leu Asn Met Glu Glu Thr Thr Ala Ala Ala 35 40 45 Ser Gln 50 204 31PRT Brassica rapa 204 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys AlaLys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe GluAla Glu Lys Ser 20 25 30 205 31 PRT Raphanus sativus 205 Ala Trp Arg LeuArg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 1 5 10 15 Lys Ala AlaAla Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser 20 25 30 206 33 PRTBrassica napus 206 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala LysAsp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu AlaGlu Lys Ser Asp 20 25 30 Thr 207 33 PRT Brassica oleracea 207 Ala TrpArg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 1 5 10 15 LysAla Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Thr208 33 PRT Brassica napus 208 Ala Trp Arg Leu Arg Ile Pro Glu Thr ThrCys Ala Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu AlaPhe Glu Ala Glu Lys Ser Asp 20 25 30 Thr 209 34 PRT Brassica rapa 209Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 1 5 1015 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 2530 Thr Thr 210 33 PRT Brassica rapa 210 Ala Trp Arg Leu Arg Ile Pro GluThr Thr Cys Ala Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala AlaLeu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Thr 211 33 PRT Brassicaoleracea 211 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp IleGln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Gly Ala Glu LysSer Asp 20 25 30 Thr 212 26 PRT Brassica juncea 212 Ala Trp Arg Leu ArgIle Ser Glu Thr Thr Cys Pro Lys Glu Ile Gln 1 5 10 15 Lys Ala Ala AlaGlu Ala Ala Val Ala Phe 20 25 213 26 PRT Brassica juncea 213 Ala Trp ArgLeu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 1 5 10 15 Lys AlaAla Ala Glu Ala Ala Val Ala Phe 20 25 214 26 PRT Brassica napus 214 AlaTrp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 1 5 10 15Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 20 25 215 26 PRT Raphanussativus 215 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Asp IleGln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 20 25 216 26 PRTArabidopsis thaliana 216 Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys AlaLys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe 20 25217 26 PRT Arabidopsis thaliana 217 Ala Trp Arg Leu Arg Ile Pro Glu SerThr Cys Ala Lys Glu Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala LeuAsn Phe 20 25 218 26 PRT Arabidopsis thaliana 218 Ala Trp Arg Leu ArgIle Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala AlaGlu Ala Ala Leu Ala Phe 20 25 219 49 PRT Brassica napus 219 Ala Glu ValAsn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 1 5 10 15 Ala ThrAla Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His GlyVal Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 35 40 45 Glu 22049 PRT Brassica napus 220 Ala Glu Val Asn Asp Thr Thr Thr Glu His GlyMet Asn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln Ala Glu ValAsn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu Glu Thr Met ValGlu Ala Val Phe Thr Gly 35 40 45 Glu 221 49 PRT Brassica rapa 221 AlaGlu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 1 5 10 15Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 35 40 45Glu 222 49 PRT Brassica napus 222 Ala Glu Val Asn Asp Thr Thr Thr GluHis Gly Met Asn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln AlaGlu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu Glu ThrMet Val Glu Ala Val Phe Thr Gly 35 40 45 Glu 223 49 PRT Brassica napus223 Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 1 510 15 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 2025 30 His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 3540 45 Glu 224 49 PRT Brassica napus 224 Ala Glu Val Asn Asp Thr Thr ThrAsp His Gly Met Asn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser GlnAla Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu GluThr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu 225 49 PRT Brassicaoleracea 225 Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met GluGlu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr ThrThr Asp 20 25 30 His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val PheThr Glu 35 40 45 Glu 226 49 PRT Brassica napus 226 Ala Glu Val Asn AspThr Thr Thr Glu His Gly Met Asn Met Glu Glu 1 5 10 15 Ala Thr Ala ValAla Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val AspMet Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu 227 49 PRTBrassica oleracea VARIANT (1)...(49) Xaa = Any Amino Acid 227 Thr GluVal Ser Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 1 5 10 15 ThrThr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Xaa Xaa Thr Asp 20 25 30 HisGly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu228 41 PRT Brassica rapa 228 Asp His Gly Met Asn Met Lys Asn Thr Thr AlaVal Ala Ser Gln Val 1 5 10 15 Glu Val Asn Asp Thr Thr Thr Asp His GlyVal Asp Met Glu Glu Thr 20 25 30 Leu Val Glu Ala Val Phe Thr Glu Glu 3540 229 41 PRT Raphanus sativus 229 Asp His Gly Met Asn Met Lys Asn ThrThr Ala Val Ala Ser Gln Val 1 5 10 15 Glu Val Asn Asp Thr Thr Thr AspHis Gly Val Asp Met Glu Glu Thr 20 25 30 Leu Val Glu Ala Val Phe Thr GluGlu 35 40 230 37 PRT Brassica napus 230 Thr Thr Asn Asp His Gly Met AsnMet Ala Ser Gln Ala Glu Val Asn 1 5 10 15 Asp Thr Thr Asp His Gly LeuAsp Met Glu Glu Thr Met Val Glu Ala 20 25 30 Val Phe Thr Glu Glu 35 23137 PRT Brassica oleracea 231 Thr Thr Asn Asp His Gly Met Asn Met Ala SerGln Ala Glu Val Asn 1 5 10 15 Asp Thr Thr Asp His Gly Leu Asp Met GluGlu Thr Met Val Glu Ala 20 25 30 Val Phe Thr Glu Glu 35 232 37 PRTBrassica napus 232 Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln ValGlu Val Asn 1 5 10 15 Asp Thr Thr Asp His Asp Leu Asp Met Glu Glu ThrIle Val Glu Ala 20 25 30 Val Phe Arg Glu Glu 35 233 37 PRT Brassica rapa233 Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn 1 510 15 Asp Thr Thr Asp His Asp Leu Asp Met Glu Glu Thr Met Val Glu Ala 2025 30 Val Phe Arg Glu Glu 35 234 44 PRT Brassica rapa 234 Thr Thr AsnAsp Arg Gly Met Asn Met Glu Glu Thr Ser Ala Val Ala 1 5 10 15 Ser ProAla Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Met 20 25 30 Glu GluThr Met Val Glu Ala Val Phe Arg Asp Glu 35 40 235 44 PRT Brassicaoleracea 235 Thr Thr Asn Asp Gln Gly Met Asn Met Glu Glu Met Thr Val ValAla 1 5 10 15 Ser Gln Ala Glu Val Ser Asp Thr Thr Thr Tyr His Gly LeuAsp Met 20 25 30 Glu Glu Thr Met Val Glu Ala Val Phe Ala Glu Glu 35 40236 26 PRT Brassica juncea 236 Gln Ala Glu Leu Asn Asp Thr Thr Ala AspHis Gly Leu Asp Val Glu 1 5 10 15 Glu Thr Ile Val Glu Ala Ile Phe ThrGlu 20 25 237 26 PRT Brassica juncea 237 Gln Ala Glu Leu Asn Asp Thr ThrAla Asp His Gly Leu Asp Val Glu 1 5 10 15 Glu Thr Ile Val Glu Ala IlePhe Thr Glu 20 25 238 26 PRT Brassica napus 238 Lys Ala Glu Ile Asn AsnThr Thr Ala Asp His Gly Ile Asp Val Glu 1 5 10 15 Glu Thr Ile Val GluAla Ile Phe Thr Glu 20 25 239 26 PRT Raphanus sativus 239 Gln Ala GluIle Asn Asp Thr Thr Thr Asp His Gly Leu Asp Ile Glu 1 5 10 15 Glu ThrIle Val Glu Ala Ile Phe Thr Glu 20 25 240 28 PRT Arabidopsis thaliana240 Gln Asp Glu Thr Cys Asp Thr Thr Thr Thr Asp His Gly Leu Asp Met 1 510 15 Glu Glu Thr Met Val Glu Ala Ile Tyr Thr Pro Glu 20 25 241 28 PRTArabidopsis thaliana 241 Gln Asp Glu Met Cys His Met Thr Thr Asp Ala HisGly Leu Asp Met 1 5 10 15 Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr ProGlu 20 25 242 27 PRT Arabidopsis thaliana 242 Gln Asp Glu Met Cys AspAla Thr Thr Asp His Gly Phe Asp Met Glu 1 5 10 15 Glu Thr Leu Val GluAla Ile Tyr Thr Ala Glu 20 25 243 49 PRT Brassica napus 243 Gln Ser GluGly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 1 5 10 15 Val ValThr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 20 25 30 Met LeuGlu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 35 40 45 Leu 24449 PRT Brassica napus 244 Gln Ser Glu Gly Phe Asn Met Ala Lys Glu SerThr Val Glu Ala Ala 1 5 10 15 Val Val Thr Glu Glu Pro Ser Lys Gly SerTyr Met Asp Glu Glu Trp 20 25 30 Met Leu Glu Met Pro Thr Leu Leu Ala AspMet Ala Glu Gly Met Leu 35 40 45 Leu 245 49 PRT Brassica rapa 245 GlnSer Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 1 5 10 15Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 20 25 30Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 35 40 45Leu 246 49 PRT Brassica napus 246 Gln Ser Glu Gly Phe Asn Met Ala LysGlu Ser Thr Val Glu Ala Ala 1 5 10 15 Val Val Thr Glu Glu Pro Ser LysGly Ser Tyr Met Asp Glu Glu Trp 20 25 30 Met Leu Glu Met Pro Thr Leu LeuAla Asp Met Ala Glu Gly Met Leu 35 40 45 Leu 247 49 PRT Brassica napus247 Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 1 510 15 Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 2025 30 Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 3540 45 Leu 248 49 PRT Brassica napus 248 Gln Ser Glu Gly Phe Asn Met AlaGlu Glu Ser Thr Val Glu Ala Ala 1 5 10 15 Val Val Thr Asp Glu Leu SerLys Gly Phe Tyr Met Asp Glu Glu Trp 20 25 30 Thr Tyr Glu Met Pro Thr LeuLeu Ala Asp Met Ala Ala Gly Met Leu 35 40 45 Leu 249 49 PRT Brassicaoleracea 249 Gln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu AlaAla 1 5 10 15 Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp GluGlu Trp 20 25 30 Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala GlyMet Leu 35 40 45 Leu 250 49 PRT Brassica napus 250 Gln Ser Glu Gly PheAsn Met Ala Glu Glu Ser Thr Val Glu Ala Ala 1 5 10 15 Val Val Thr AspGlu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 20 25 30 Thr Tyr Glu MetPro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 35 40 45 Leu 251 49 PRTBrassica oleracea 251 Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser ThrAla Glu Ala Ala 1 5 10 15 Val Val Thr Glu Glu Leu Ser Lys Gly Val TyrMet Asp Glu Glu Trp 20 25 30 Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp MetAla Ala Gly Met Leu 35 40 45 Leu 252 48 PRT Brassica rapa 252 Gln ArgGlu Gly Phe Tyr Met Thr Glu Glu Thr Arg Val Glu Gly Val 1 5 10 15 ValThr Glu Glu Gln Asn Asn Trp Phe Tyr Met Asp Glu Glu Trp Met 20 25 30 PheGly Met Pro Thr Leu Leu Val Asp Met Ala Glu Gly Met Leu Ile 35 40 45 25348 PRT Raphanus sativus 253 Gln Arg Glu Gly Phe Tyr Met Thr Glu Glu ThrArg Val Glu Gly Val 1 5 10 15 Val Thr Glu Glu Gln Asn Asn Trp Phe TyrMet Asp Glu Glu Trp Met 20 25 30 Phe Gly Met Pro Thr Leu Leu Val Asp MetAla Glu Gly Met Leu Leu 35 40 45 254 49 PRT Brassica napus 254 Gln ArgAsp Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Val 1 5 10 15 ValPro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 20 25 30 MetPhe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 35 40 45 Leu255 49 PRT Brassica oleracea 255 Gln Arg Asp Gly Phe Tyr Met Ala Glu GluThr Thr Val Glu Gly Val 1 5 10 15 Val Pro Glu Glu Gln Met Ser Lys GlyPhe Tyr Met Asp Glu Glu Trp 20 25 30 Met Phe Gly Met Pro Thr Leu Leu AlaAsp Met Ala Ala Gly Met Leu 35 40 45 Leu 256 49 PRT Brassica napus 256Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Val Gly Val 1 5 1015 Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 20 2530 Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 35 4045 Leu 257 49 PRT Brassica rapa 257 Gln Arg Glu Gly Phe Tyr Met Ala GluGlu Thr Thr Val Glu Gly Ile 1 5 10 15 Val Pro Glu Glu Gln Met Ser LysGly Phe Tyr Met Asp Glu Glu Trp 20 25 30 Met Phe Gly Met Pro Thr Leu LeuAla Asp Met Ala Ala Gly Met Leu 35 40 45 Leu 258 49 PRT Brassica rapa258 Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Val 1 510 15 Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 2025 30 Thr Phe Glu Met Pro Arg Leu Leu Ala Asp Met Ala Glu Gly Met Leu 3540 45 Leu 259 48 PRT Brassica oleracea 259 Gln Arg Glu Gly Phe Tyr LeuAla Glu Glu Thr Thr Val Glu Gly Val 1 5 10 15 Val Thr Glu Glu Gln SerLys Gly Phe Tyr Met Asp Glu Glu Trp Thr 20 25 30 Phe Gly Met Gln Ser PheLeu Ala Asp Met Ala Glu Gly Met Leu Phe 35 40 45 260 29 PRT Brassicajuncea 260 Glu Ser Ser Glu Gly Phe Tyr Met Asp Glu Glu Phe Met Phe GlyMet 1 5 10 15 Pro Thr Leu Trp Ala Ser Met Ala Glu Gly Met Leu Leu 20 25261 29 PRT Brassica juncea 261 Glu Ser Ser Glu Gly Phe Tyr Met Ala GluGlu Phe Met Phe Gly Met 1 5 10 15 Pro Thr Leu Trp Ala Ser Val Ala GluGly Met Leu Leu 20 25 262 30 PRT Brassica napus 262 Glu Asn Asn Asp GlyPhe Tyr Met Asp Glu Glu Glu Ser Met Phe Gly 1 5 10 15 Met Pro Ala LeuLeu Ala Ser Met Ala Glu Gly Met Leu Leu 20 25 30 263 29 PRT Raphanussativus 263 Val Asn Asn Asp Glu Phe Tyr Met Asp Glu Glu Ser Met Phe GlyMet 1 5 10 15 Pro Ser Leu Leu Ala Ser Met Ala Glu Gly Met Leu Leu 20 25264 29 PRT Arabidopsis thaliana 264 Gln Ser Glu Gly Ala Phe Tyr Met AspGlu Glu Thr Met Phe Gly Met 1 5 10 15 Pro Thr Leu Leu Asp Asn Met AlaGlu Gly Met Leu Leu 20 25 265 29 PRT Arabidopsis thaliana 265 Gln SerGln Asp Ala Phe Tyr Met Asp Glu Glu Ala Met Leu Gly Met 1 5 10 15 SerSer Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu 20 25 266 29 PRTArabidopsis thaliana 266 Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu AlaMet Phe Glu Met 1 5 10 15 Pro Ser Leu Leu Ala Asn Met Ala Glu Gly MetLeu Leu 20 25 267 50 PRT Brassica napus 267 Ala Trp Arg Leu Arg Ile ProGlu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu AlaAla Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Gln Asn GlyLeu Asn Met Glu Glu Thr Thr Ala Val Ala 35 40 45 Ser Gln 50 268 50 PRTBrassica napus 268 Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His LysAsp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu AlaGlu Lys Ser Asp 20 25 30 Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu ThrThr Ala Val Ala 35 40 45 Ser Gln 50 269 50 PRT Brassica rapa 269 Ala TrpArg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 LysAla Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 ValThr Met Gln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala 35 40 45 SerGln 50 270 50 PRT Brassica napus 270 Ala Trp Arg Leu Arg Ile Pro Glu ThrThr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala LeuAla Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Gln Asn Gly Gln AsnMet Glu Glu Thr Thr Ala Val Ala 35 40 45 Ser Gln 50 271 44 PRT Brassicanapus 271 Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp IleGln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu LysSer Asp 20 25 30 Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 35 40272 44 PRT Brassica napus 272 Ala Ser Arg Leu Arg Ile Pro Glu Thr ThrCys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu Ala Ala Leu AlaPhe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met Glu Glu Thr Met Ala ValAla Ser Gln 35 40 273 44 PRT Brassica oleracea 273 Ala Trp Arg Leu ArgIle Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala AlaGlu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Val Thr Met GluGlu Thr Met Ala Val Ala Ser Gln 35 40 274 50 PRT Brassica napus 274 AlaTrp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 35 40 45Ser Gln 50 275 50 PRT Brassica oleracea 275 Ala Trp Arg Leu Arg Ile ProGlu Thr Thr Cys His Lys Asp Ile Gln 1 5 10 15 Lys Ala Ala Ala Glu AlaAla Leu Ala Phe Glu Ala Glu Lys Ser Asp 20 25 30 Ala Thr Met Gln Asn GlyLeu Asn Met Glu Glu Thr Thr Ala Ala Ala 35 40 45 Ser Gln 50 276 50 PRTBrassica napus 276 Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met AsnMet Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn AspThr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu Glu Thr Met Val Glu AlaVal Phe Thr Gly 35 40 45 Glu Gln 50 277 50 PRT Brassica napus 277 AlaGlu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 1 5 10 15Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 35 40 45Glu Gln 50 278 50 PRT Brassica rapa 278 Ala Glu Val Asn Asp Thr Thr ThrGlu His Gly Met Asn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser GlnAla Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu GluThr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu Gln 50 279 50 PRTBrassica napus 279 Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met AsnMet Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn AspThr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu Glu Thr Met Val Glu AlaVal Phe Thr Gly 35 40 45 Glu Gln 50 280 50 PRT Brassica napus 280 AlaGlu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 1 5 10 15Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 35 40 45Glu Gln 50 281 50 PRT Brassica napus 281 Ala Glu Val Asn Asp Thr Thr ThrAsp His Gly Met Asn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser GlnAla Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu GluThr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu Gln 50 282 50 PRTBrassica oleracea 282 Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly MetAsn Met Glu Glu 1 5 10 15 Ala Thr Ala Val Ala Ser Gln Ala Glu Val AsnAsp Thr Thr Thr Asp 20 25 30 His Gly Val Asp Met Glu Glu Thr Met Val GluAla Val Phe Thr Glu 35 40 45 Glu Gln 50 283 50 PRT Brassica napus 283Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 1 5 1015 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 2530 His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 35 4045 Glu Gln 50 284 50 PRT Brassica oleracea 284 Thr Glu Val Ser Asp ThrThr Thr Asp His Gly Met Asn Met Glu Glu 1 5 10 15 Thr Thr Ala Val AlaSer Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 20 25 30 His Gly Val Asp MetGlu Glu Thr Met Val Glu Ala Val Phe Thr Glu 35 40 45 Glu Gln 50 285 50PRT Brassica napus 285 Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr ValGlu Ala Ala Val 1 5 10 15 Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr MetAsp Glu Glu Trp Met 20 25 30 Leu Glu Met Pro Thr Leu Leu Ala Asp Met AlaGlu Gly Met Leu Leu 35 40 45 Pro Pro 50 286 50 PRT Brassica rapa 286 SerGlu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 1 5 10 15Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 20 25 30Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 35 40 45Pro Pro 50 287 50 PRT Brassica napus 287 Ser Glu Gly Phe Asn Met Ala LysGlu Ser Thr Val Glu Ala Ala Val 1 5 10 15 Val Thr Glu Glu Pro Ser LysGly Ser Tyr Met Asp Glu Glu Trp Met 20 25 30 Leu Glu Met Pro Thr Leu LeuAla Asp Met Ala Glu Gly Met Leu Leu 35 40 45 Pro Pro 50 288 48 PRTBrassica napus 288 Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val GluAla Ala Val 1 5 10 15 Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met AspGlu Glu Trp Met 20 25 30 Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala GluGly Met Leu Leu 35 40 45 289 50 PRT Brassica napus 289 Ser Glu Gly PheAsn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 1 5 10 15 Val Thr GluGlu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 20 25 30 Leu Glu MetPro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 35 40 45 Pro Pro 50290 50 PRT Brassica napus 290 Ser Glu Gly Phe Asn Met Ala Glu Glu SerThr Val Glu Ala Ala Val 1 5 10 15 Val Thr Asp Glu Leu Ser Lys Gly PheTyr Met Asp Glu Glu Trp Thr 20 25 30 Tyr Glu Met Pro Thr Leu Leu Ala AspMet Ala Ala Gly Met Leu Leu 35 40 45 Pro Pro 50 291 50 PRT Brassicaoleracea 291 Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala AlaVal 1 5 10 15 Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu GluTrp Thr 20 25 30 Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly MetLeu Leu 35 40 45 Pro Pro 50 292 50 PRT Brassica napus 292 Ser Glu GlyPhe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val 1 5 10 15 Val ThrAsp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr 20 25 30 Tyr GluMet Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu 35 40 45 Pro Pro50 293 50 PRT Brassica oleracea 293 Ser Glu Gly Phe Asn Met Ala Lys GluSer Thr Ala Glu Ala Ala Val 1 5 10 15 Val Thr Glu Glu Leu Ser Lys GlyVal Tyr Met Asp Glu Glu Trp Thr 20 25 30 Tyr Glu Met Pro Thr Leu Leu AlaAsp Met Ala Ala Gly Met Leu Leu 35 40 45 Pro Pro 50 294 205 PRT Medicagotruncatula G3362 polypeptide 294 Met Phe Thr Met Asn Gln Phe Ser Glu SerHis Asp Pro Cys Ser Ser 1 5 10 15 Ser Ser Glu Arg Phe Leu Ala Glu ThrMet Pro Lys Lys Arg Ala Gly 20 25 30 Arg Lys Lys Phe Arg Glu Thr Arg HisPro Val Tyr Arg Gly Val Arg 35 40 45 Lys Arg Asp Ser Gly Lys Trp Val CysGlu Val Arg Glu Pro Asn Lys 50 55 60 Lys Thr Arg Ile Trp Leu Gly Thr PhePro Thr Pro Glu Met Ala Ala 65 70 75 80 Arg Ala His Asp Val Ala Ala IleAla Leu Arg Gly Arg Ser Ala Cys 85 90 95 Leu Asn Phe Ala Asp Ser Ala TrpLys Leu Pro Val Pro Ala Thr Ser 100 105 110 Glu Ala Arg Asp Ile Gln LysAla Ala Ala Glu Ala Ala Glu Ala Phe 115 120 125 Arg Pro Glu Ser Val PheGlu Asn Ser Glu Glu Arg Lys Asp Ser Glu 130 135 140 Pro Ser Ser Thr ValAla Val Ser Glu Thr Val Met Glu Gln Arg Glu 145 150 155 160 Glu Glu GluAsp Thr Val Pro Glu Tyr Leu Arg Asn Met Val Leu Met 165 170 175 Ser ProAla His Tyr Trp Gly Ser Asp Cys Gly Val Ala Asp Val Glu 180 185 190 PheAsp Glu Thr Glu Val Ser Leu Trp Ser Tyr Ser Phe 195 200 205 295 215 PRTMedicago truncatula G3364 polypeptide 295 Met Phe Thr Thr Asn Asn SerSer Tyr Ser His Ser Ile Ser Ser Glu 1 5 10 15 Ala Ser Ser Ser Tyr TyrAsn Ser Leu Pro Glu Ser Glu Ile Arg Leu 20 25 30 Ala Ala Ser Asn Pro LysLys Arg Ala Gly Arg Lys Ile Phe Lys Glu 35 40 45 Thr Arg His Pro Val TyrArg Gly Val Arg Lys Arg Asn Leu Asp Lys 50 55 60 Trp Val Cys Glu Met ArgGlu Pro Asn Thr Lys Thr Arg Ile Trp Leu 65 70 75 80 Gly Thr Phe Pro ThrAla Glu Met Ala Ala Arg Ala His Asp Val Ala 85 90 95 Ala Met Ala Leu ArgGly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser 100 105 110 Val Trp Arg LeuPro Ile Pro Ala Thr Ser Ser Ile Lys Asp Ile Gln 115 120 125 Lys Ala AlaThr Lys Ala Ala Glu Ala Phe Arg Pro Asp Asn Thr Ile 130 135 140 Met IleThr Asn Ile Glu Thr Val Val Ala Val Val Ala Thr Lys Glu 145 150 155 160Leu Asn Met Phe Cys Val Glu Glu Glu Glu Glu Met Leu Asn Met Pro 165 170175 Glu Phe Trp Arg Asn Met Ala Leu Met Ser Pro Thr His Ser Phe Glu 180185 190 Tyr His Asp Gln Tyr Glu Asp Phe His Phe Gln Asp Phe Gln Asp Asp195 200 205 Glu Val Ser Leu Trp Asn Phe 210 215 296 220 PRT Medicagotruncatula G3365 polypeptide 296 Met Lys Ser Ser Leu Asp Glu Ser Ser TyrVal Glu Asn Asn Ser Ser 1 5 10 15 Ser Ser Glu Ile Leu Leu Ala Ser GluGln Pro Lys Lys Arg Ala Gly 20 25 30 Arg Arg Lys Phe Lys Glu Thr Arg HisPro Val Tyr Arg Gly Val Arg 35 40 45 Arg Arg Asn Asn Asn Asn Asn Lys TrpVal Cys Glu Val Arg Val Pro 50 55 60 Asn Asp Lys Ser Thr Arg Ile Trp LeuGly Thr Tyr Pro Thr Pro Glu 65 70 75 80 Met Ala Ala Arg Ala His Asp ValAla Ala Leu Ala Leu Arg Gly Lys 85 90 95 Ser Ala Cys Leu Asn Phe Ala AspSer Ala Trp Arg Leu Ala Leu Pro 100 105 110 Ala Thr Asn Asn Ala Lys GluIle Arg Lys Met Ala Ala Glu Ala Ala 115 120 125 Leu Ala Phe Ala Val ValAla Asp Ser Lys Glu Gln Thr Met Ile Ser 130 135 140 Asn Cys Asp Val AsnSer Val Gly Val Met Glu Val Asp Asn Lys Pro 145 150 155 160 Leu Gln GlyLeu Cys Val Glu Val Pro Glu Glu Glu Met Leu His Asp 165 170 175 Trp PheArg Ser Met Ala Asp Glu Pro Leu Arg Ser Pro Ile Thr Pro 180 185 190 PheIle Arg His Gly Arg Asp Gln Trp Asn Asn Val Asp Ile Asp Gln 195 200 205Val Asp Ala Glu Val Ser Leu Trp Asn Phe Thr Ile 210 215 220 297 227 PRTMedicago truncatula G3366 polypeptide 297 Met Tyr Pro Thr Thr Asn SerVal Ser Ser Ser Ser Ser Asp Met Ser 1 5 10 15 Leu Pro Asn Ser Glu GlySer His Trp Met Ser Ile Cys Asn Glu Glu 20 25 30 Met Arg Leu Ala Ala ThrThr Pro Lys Lys Arg Ala Gly Arg Lys Lys 35 40 45 Phe Lys Glu Thr Arg HisPro Val Tyr Arg Gly Val Arg Lys Arg Asn 50 55 60 Leu Asp Lys Trp Val CysGlu Met Arg Glu Pro Asn Lys Lys Thr Lys 65 70 75 80 Ile Trp Leu Gly ThrPhe Pro Thr Ala Glu Met Ala Ala Arg Ala His 85 90 95 Asp Val Ala Ala MetAla Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe 100 105 110 Ala Asp Ser AlaTrp Arg Leu Pro Lys Pro Ala Thr Thr Gln Ala Lys 115 120 125 Asp Ile GlnLys Ala Ala Thr Glu Ala Ala Glu Ala Phe Arg Pro Asp 130 135 140 Lys ThrLeu Leu Thr Asn His Asn Asp Asn Asp Asn Asp Asn Asp Lys 145 150 155 160Glu Asn Asp Met Ala Val Val Ala Thr Ala Thr Glu Glu Gln Ser Met 165 170175 Ile Cys Met Glu Glu Lys Glu Glu Gly Val Met Asn Met Gln Glu Met 180185 190 Trp Ser Asn Met Ala Leu Met Ser Pro Thr His Ser Leu Gly Tyr Tyr195 200 205 Glu Tyr Gln Tyr Ile Asn Glu Asp Phe Gln Asp Glu Glu Val SerLeu 210 215 220 Trp Ser Phe 225 298 248 PRT Medicago truncatula G3367polypeptide 298 Met Ile Ser Thr Asn Asn Ser Ser Tyr Ser His Ser Ile SerSer Lys 1 5 10 15 Asp Phe Ser Pro Phe Asp Ala Ser Ser Pro Asp Ser GluVal Arg Leu 20 25 30 Ala Ser Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys IlePhe Lys Glu 35 40 45 Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg AsnLeu Asn Lys 50 55 60 Trp Val Cys Glu Met Arg Glu Pro Asn Thr Lys Asn ArgIle Trp Leu 65 70 75 80 Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg AlaHis Asp Val Ala 85 90 95 Ala Ile Ala Leu Arg Gly Arg Tyr Ala Cys Leu AsnPhe Ala Asp Ser 100 105 110 Val Trp Arg Leu Pro Ile Pro Ala Thr Ser AlaIle Lys Asp Ile Gln 115 120 125 Lys Ala Ala Thr Lys Ala Ala Glu Ala PheArg Pro Asp Asn Thr Leu 130 135 140 Met Thr Ser Asp Ile Asp Thr Val ValAla Val Val Ala Thr Gln Glu 145 150 155 160 Leu Asn Met Phe Arg Val GluVal Glu Glu Glu Glu Val Leu Asn Met 165 170 175 Pro Glu Leu Trp Arg AsnMet Ala Leu Met Ser Pro Thr His Ser Phe 180 185 190 Gly Tyr His Asp GlnTyr Glu Asp Ile His Ile Gln Asp Phe Gln Asp 195 200 205 Asp Glu Asp PheLys Lys Arg Ser Val Thr Thr Ile Trp Ala Val Thr 210 215 220 Ser Ile GlyVal His Ser Leu His Phe Thr Val Ile Ser Arg Ile Val 225 230 235 240 MetArg Thr Leu Leu Leu Cys Val 245 299 229 PRT Medicago truncatula G3368polypeptide 299 Met Asp Phe Phe Met Ser Ser Phe Ser Asp Tyr Ser Asp ThrSer Ser 1 5 10 15 Ser Glu Thr Ala Ser Ser Asn Arg Thr Ser Ser Ser GluVal Ile Leu 20 25 30 Ala Pro Ala Arg Pro Lys Lys Arg Ala Gly Arg Arg ValPhe Lys Glu 35 40 45 Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg Arg LysAsn Asn Lys 50 55 60 Trp Val Cys Glu Met Arg Val Pro Asn Asn Ile Val AsnLys Asn Asn 65 70 75 80 Lys Ser Arg Ile Trp Leu Gly Thr Tyr Pro Thr ProGlu Met Ala Ala 85 90 95 Arg Ala His Asp Val Ala Ala Leu Thr Leu Lys GlyLys Ser Ala Cys 100 105 110 Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu ArgLeu Pro Glu Ser Asn 115 120 125 Asp Ala Thr Glu Ile Arg Arg Ala Ala MetGlu Ala Ala Gln Leu Phe 130 135 140 Ala Val Glu Asp Lys Gln Cys Cys ValThr Val Glu Asp Gly Val Phe 145 150 155 160 Met Asp Met Glu Asp Ser LysAsn Met Leu Glu Ala Gln Val Pro Val 165 170 175 Val Ser Ser Glu Phe GluAsp Met His His Leu Leu Leu Ser Ile Ala 180 185 190 Asn Glu Pro Leu ArgSer Ala Pro Pro Ser Pro Thr Asn Tyr Gly Ser 195 200 205 Tyr Asn Trp GlyAsp Met Glu Ile Phe Asp Thr Gln Leu Val Ser Leu 210 215 220 Trp Asn PheSer Ile 225 300 260 PRT Medicago truncatula G3369 polypeptide 300 MetAsp Met Phe Thr Asn Asn Asn Ser Tyr Ser His Pro Phe Ser Pro 1 5 10 15Thr Cys Ser Glu Ser Ser Phe Pro Asn Ser Glu Gly Ser Gln Gly Met 20 25 30Ser Ile Ser Asn Glu Glu Val Arg Leu Ala Ala Thr Thr Pro Lys Lys 35 40 45Arg Ala Gly Arg Lys Lys Phe Lys Glu Thr Arg His Pro Val Tyr Arg 50 55 60Gly Val Arg Lys Arg Asn Leu Asp Lys Trp Val Cys Glu Met Arg Glu 65 70 7580 Pro Asn Lys Lys Thr Lys Ile Trp Leu Gly Thr Phe Pro Thr Ala Glu 85 9095 Met Ala Ala Arg Ala His Asp Val Ala Ala Met Ala Leu Arg Gly Arg 100105 110 Tyr Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Pro Ile Pro115 120 125 Ala Thr Thr Gln Ala Lys Asp Ile Gln Lys Ala Ala Ala Gln AlaAla 130 135 140 Glu Ala Phe Arg Pro Asp Lys Thr Ser Ile Thr Asn Asp IleAsp Thr 145 150 155 160 Ala Ile Ser Thr Ser Ala Thr Ala Glu Gln Ser ArgThr Phe Met Glu 165 170 175 Glu Glu Glu Glu Gly Val Met Asn Met Pro GluLeu Leu Arg Asn Met 180 185 190 Ala Leu Met Ser Pro Thr His Ser Ser GlyTyr Asn Glu Tyr Glu Asn 195 200 205 Ile His Val Gln Asp Phe Gln Asp LeuGln Asp Phe Gln Asp Glu Glu 210 215 220 Val Leu Ile Lys His Lys Val LeuLeu Ile Pro Ser Ile Ser Ile Tyr 225 230 235 240 Glu Arg Arg Ile Glu ValTrp Tyr Val Lys Ile Ser Val Asn Phe Ile 245 250 255 Ser Tyr Leu Asn 260301 218 PRT Oryza sativa G3370 polypeptide 301 Met Glu Val Glu Glu AlaAla Tyr Arg Thr Val Trp Ser Glu Pro Pro 1 5 10 15 Lys Arg Pro Ala GlyArg Thr Lys Phe Arg Glu Thr Arg His Pro Val 20 25 30 Tyr Arg Gly Val ArgArg Arg Gly Gly Arg Pro Gly Ala Ala Gly Arg 35 40 45 Trp Val Cys Glu ValArg Val Pro Gly Ala Arg Gly Ser Arg Leu Trp 50 55 60 Leu Gly Thr Phe AlaThr Ala Glu Ala Ala Ala Arg Ala His Asp Ala 65 70 75 80 Ala Ala Leu AlaLeu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp 85 90 95 Ser Ala Trp ArgMet Pro Pro Val Pro Ala Ser Ala Ala Leu Ala Gly 100 105 110 Ala Arg GlyVal Arg Asp Ala Val Ala Val Ala Val Glu Ala Phe Gln 115 120 125 Arg GlnSer Ala Ala Pro Ser Ser Pro Ala Glu Thr Phe Ala Asp Asp 130 135 140 GlyAsp Glu Glu Glu Asp Asn Lys Asp Val Leu Pro Val Ala Ala Ala 145 150 155160 Glu Val Phe Asp Ala Gly Ala Phe Glu Leu Asp Asp Gly Phe Arg Phe 165170 175 Gly Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Leu180 185 190 Leu Val Glu Pro Pro Ala Ala Gly Ala Trp Trp Glu Asp Gly GluLeu 195 200 205 Ala Gly Ser Asp Met Pro Leu Trp Ser Tyr 210 215 302 253PRT Oryza sativa G3371 polypeptide 302 Met Glu Lys Asn Thr Ala Ala SerGly Gln Leu Met Thr Ser Ser Ala 1 5 10 15 Glu Ala Thr Pro Ser Ser ProLys Arg Pro Ala Gly Arg Thr Lys Phe 20 25 30 Gln Glu Thr Arg His Leu ValPhe Arg Gly Val Arg Trp Arg Gly Cys 35 40 45 Ala Gly Arg Trp Val Cys LysVal Arg Val Pro Gly Ser Arg Gly Asp 50 55 60 Arg Phe Trp Ile Gly Thr SerAsp Thr Ala Glu Glu Thr Ala Arg Thr 65 70 75 80 His Asp Ala Ala Met LeuAla Leu Cys Gly Ala Ser Ala Ser Leu Asn 85 90 95 Phe Ala Asp Ser Ala TrpLeu Leu His Val Pro Arg Ala Pro Val Val 100 105 110 Ser Gly Leu Arg ProPro Ala Ala Arg Cys Ala Thr Arg Cys Leu Gln 115 120 125 Gly His Arg ArgVal Pro Ala Pro Gly Arg Gly Ser Asn Ala Thr Ala 130 135 140 Thr Ala ThrSer Gly Asp Ala Ala Ser Thr Ala Pro Pro Ser Ala Pro 145 150 155 160 ValLeu Ser Ala Lys Gln Cys Glu Phe Ile Phe Leu Ser Ser Leu Asp 165 170 175Cys Trp Met Leu Met Ser Lys Leu Ile Ser Ser Ser Arg Ala Lys Gly 180 185190 Ser Leu Cys Leu Arg Lys Asn Pro Ile Ser Phe Cys Met Val Thr Asn 195200 205 Ser Tyr Thr Ala Leu Leu Leu Glu Tyr Ile Ile Leu Gln Met Asn Ser210 215 220 Met Ile Val Leu Ile His Glu Leu Ser Lys Tyr Gln Val Phe LeuLeu 225 230 235 240 Leu Thr Met Ile Thr His His Leu Phe Gln Trp Arg Arg245 250 303 214 PRT Oryza sativa G3372 polypeptide 303 Met Glu Tyr TyrGlu Gln Glu Glu Tyr Ala Thr Val Thr Ser Ala Pro 1 5 10 15 Pro Lys ArgPro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro 20 25 30 Val Tyr ArgGly Val Arg Arg Arg Gly Pro Ala Gly Arg Trp Val Cys 35 40 45 Glu Val ArgGlu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe 50 55 60 Ala Thr AlaGlu Ala Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 65 70 75 80 Leu ArgGly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Arg Leu 85 90 95 Leu ArgVal Asp Pro Ala Thr Leu Ala Thr Pro Asp Asp Ile Arg Arg 100 105 110 AlaAla Ile Glu Leu Ala Glu Ser Cys Pro His Asp Ala Ala Ala Ala 115 120 125Ala Ala Ser Ser Ser Ala Ala Ala Val Glu Ala Ser Ala Ala Ala Ala 130 135140 Pro Ala Met Met Met Gln Tyr Gln Asp Asp Met Ala Ala Thr Pro Ser 145150 155 160 Ser Tyr Asp Tyr Ala Tyr Tyr Gly Asn Met Asp Phe Asp Gln ProSer 165 170 175 Tyr Tyr Tyr Asp Gly Met Gly Gly Gly Gly Glu Tyr Gln SerTrp Gln 180 185 190 Met Asp Gly Asp Asp Asp Gly Gly Ala Gly Gly Tyr GlyGly Gly Asp 195 200 205 Val Thr Leu Trp Ser Tyr 210 304 219 PRT Oryzasativa G3373 polypeptide 304 Met Asp Thr Glu Asp Thr Ser Ser Ala Ser SerSer Ser Val Ser Pro 1 5 10 15 Pro Ser Ser Pro Gly Gly Gly His His HisArg Leu Pro Pro Lys Arg 20 25 30 Arg Ala Gly Arg Lys Lys Phe Arg Glu ThrArg His Pro Val Tyr Arg 35 40 45 Gly Val Arg Ala Arg Ala Gly Gly Ser ArgTrp Val Cys Glu Val Arg 50 55 60 Glu Pro Gln Ala Gln Ala Arg Ile Trp LeuGly Thr Tyr Pro Thr Pro 65 70 75 80 Glu Met Ala Ala Arg Ala His Asp ValAla Ala Ile Ala Leu Arg Gly 85 90 95 Glu Arg Gly Ala Glu Leu Asn Phe ProAsp Ser Pro Ser Thr Leu Pro 100 105 110 Arg Ala Arg Thr Ala Ser Pro GluAsp Ile Arg Leu Ala Ala Ala Gln 115 120 125 Ala Ala Glu Leu Tyr Arg ArgPro Pro Pro Pro Leu Ala Leu Pro Glu 130 135 140 Asp Pro Gln Glu Gly ThrSer Gly Gly Gly Ala Thr Ala Thr Ser Gly 145 150 155 160 Arg Pro Ala AlaVal Phe Val Asp Glu Asp Ala Ile Phe Asp Met Pro 165 170 175 Gly Leu IleAsp Asp Met Ala Arg Gly Met Met Leu Thr Pro Pro Ala 180 185 190 Ile GlyArg Ser Leu Asp Asp Trp Ala Ala Ile Asp Asp Asp Asp Asp 195 200 205 HisTyr His Met Asp Tyr Lys Leu Trp Met Asp 210 215 305 251 PRT Oryza sativaG3374 polypeptide 305 Met Cys Thr Ser Lys Leu Glu Glu Ile Thr Gly GluTrp Pro Pro Pro 1 5 10 15 Ala Leu Gln Ala Ala Ser Thr Thr Ser Ser SerGlu Pro Cys Arg Arg 20 25 30 Leu Ser Pro Pro Ser Ser Lys Arg Pro Ala GlyArg Thr Lys Phe His 35 40 45 Glu Thr Arg His Pro Val Phe Arg Gly Val ArgArg Arg Gly Arg Ala 50 55 60 Gly Arg Trp Val Cys Glu Val Arg Val Pro GlyArg Arg Gly Cys Arg 65 70 75 80 Leu Trp Leu Gly Thr Phe Asp Ala Ala AspAla Ala Ala Arg Ala His 85 90 95 Asp Ala Ala Met Leu Ala Leu Arg Gly ArgAla Ala Ala Cys Leu Asn 100 105 110 Phe Ala Asp Ser Ala Trp Leu Leu AlaVal Pro Pro Pro Ala Thr Leu 115 120 125 Arg Cys Ala Ala Asp Val Gln ArgAla Val Ala Arg Ala Leu Glu Asp 130 135 140 Phe Glu Gln Arg Glu Ser SerSer Ser Val Phe Pro Leu Ala Ile Asp 145 150 155 160 Val Val Ala Glu AspAla Met Ser Ala Thr Ser Glu Pro Ser Ala Ala 165 170 175 Ser Asp Asp AspAla Val Thr Ser Ser Ser Ser Thr Thr Asp Ala Asp 180 185 190 Glu Glu AlaSer Pro Phe Glu Leu Asp Val Val Ser Asp Met Gly Trp 195 200 205 Ser LeuTyr Tyr Ala Ser Leu Ala Glu Gly Leu Leu Met Glu Pro Pro 210 215 220 AlaSer Gly Ala Ser Ser Asp Asp Asp Asp Asp Ala Ile Val Asp Ser 225 230 235240 Gly Asp Ile Ala Asp Val Ser Leu Trp Ser Tyr 245 250 306 219 PRTOryza sativa G3375 polypeptide 306 Met Glu Trp Ala Tyr Tyr Gly Ser GlyTyr Ser Ser Ser Gly Thr Pro 1 5 10 15 Ser Pro Val Gly Gly Asp Gly AspGlu Asp Ser Tyr Met Thr Val Ser 20 25 30 Ser Ala Pro Pro Lys Arg Arg AlaGly Arg Thr Lys Phe Lys Glu Thr 35 40 45 Arg His Pro Val Tyr Lys Gly ValArg Ser Arg Asn Pro Gly Arg Trp 50 55 60 Val Cys Glu Val Arg Glu Pro HisGly Lys Gln Arg Ile Trp Leu Gly 65 70 75 80 Thr Phe Glu Thr Ala Glu MetAla Ala Arg Ala His Asp Val Ala Ala 85 90 95 Met Ala Leu Arg Gly Arg AlaAla Cys Leu Asn Phe Ala Asp Ser Pro 100 105 110 Arg Arg Leu Arg Val ProPro Leu Gly Ala Gly His Glu Glu Ile Arg 115 120 125 Arg Ala Ala Val GluAla Ala Glu Leu Phe Arg Pro Ala Pro Gly Gln 130 135 140 His Asn Ala AlaAla Glu Ala Ala Ala Ala Val Ala Ala Gln Ala Thr 145 150 155 160 Ala AlaSer Ala Glu Leu Phe Ala Asp Phe Pro Cys Tyr Pro Met Asp 165 170 175 GlyLeu Glu Phe Glu Met Gln Gly Tyr Leu Asp Met Ala Gln Gly Met 180 185 190Leu Ile Glu Pro Pro Pro Leu Ala Gly Gln Ser Thr Trp Ala Glu Glu 195 200205 Asp Tyr Asp Cys Glu Val Asn Leu Trp Ser Tyr 210 215 307 224 PRTOryza sativa G3376 polypeptide 307 Met Asp Val Ser Ala Ala Leu Ser SerAsp Tyr Ser Ser Gly Thr Pro 1 5 10 15 Ser Pro Val Ala Ala Asp Ala AspAsp Gly Ser Ser Ala Tyr Met Thr 20 25 30 Val Ser Ser Ala Pro Pro Lys ArgArg Ala Gly Arg Thr Lys Phe Lys 35 40 45 Glu Thr Arg His Pro Val Phe LysGly Val Arg Arg Arg Asn Pro Gly 50 55 60 Arg Trp Val Cys Glu Val Arg GluPro His Gly Lys Gln Arg Ile Trp 65 70 75 80 Leu Gly Thr Phe Glu Thr AlaGlu Met Ala Ala Arg Ala His Asp Val 85 90 95 Ala Ala Leu Ala Leu Arg GlyArg Ala Ala Cys Leu Asn Phe Ala Asp 100 105 110 Ser Pro Arg Arg Leu ArgVal Pro Pro Ile Gly Ala Ser His Asp Asp 115 120 125 Ile Arg Arg Ala AlaAla Glu Ala Ala Glu Ala Phe Arg Pro Pro Pro 130 135 140 Asp Glu Ser AsnAla Ala Thr Glu Val Ala Ala Ala Ala Ser Gly Ala 145 150 155 160 Thr AsnSer Asn Ala Glu Gln Phe Ala Ser His Pro Tyr Tyr Glu Val 165 170 175 MetAsp Asp Gly Leu Asp Leu Gly Met Gln Gly Tyr Leu Asp Met Ala 180 185 190Gln Gly Met Leu Ile Asp Pro Pro Pro Met Ala Cys Asp Pro Ala Val 195 200205 Gly Gly Gly Glu Asp Asp Asn Asp Gly Glu Val Gln Leu Trp Ser Tyr 210215 220 308 236 PRT Oryza sativa G3377 polypeptide 308 Met Glu Lys AsnThr Thr Ala Met Gly Gln Leu Met Ser Ser Ser Ala 1 5 10 15 Thr Thr AlaAla Thr Ala Thr Gly Pro Ala Ser Pro Lys Arg Pro Ala 20 25 30 Gly Arg ThrLys Phe Gln Glu Thr Arg His Pro Val Phe Arg Gly Val 35 40 45 Arg Arg ArgGly Arg Ala Gly Arg Trp Val Cys Glu Val Arg Val Pro 50 55 60 Gly Ser ArgGly Asp Arg Leu Trp Val Gly Thr Phe Asp Thr Ala Glu 65 70 75 80 Glu AlaAla Arg Ala His Asp Ala Ala Met Leu Ala Leu Cys Gly Ala 85 90 95 Ser AlaSer Leu Asn Phe Ala Asp Ser Ala Trp Leu Leu His Val Pro 100 105 110 ArgAla Pro Val Ala Ser Gly His Asp Gln Leu Pro Asp Val Gln Arg 115 120 125Ala Ala Ser Glu Ala Val Ala Glu Phe Gln Arg Arg Gly Ser Thr Ala 130 135140 Ala Thr Ala Thr Ala Thr Ser Gly Asp Ala Ala Ser Thr Ala Pro Pro 145150 155 160 Ser Ser Ser Pro Val Leu Ser Pro Asn Asp Asp Asn Ala Ser SerAla 165 170 175 Ser Thr Pro Ala Val Ala Ala Ala Leu Asp His Gly Asp MetPhe Gly 180 185 190 Gly Met Arg Thr Asp Leu Tyr Phe Ala Ser Leu Ala GlnGly Leu Leu 195 200 205 Ile Glu Pro Pro Pro Pro Pro Thr Thr Ala Glu GlyPhe Cys Asp Asp 210 215 220 Glu Gly Cys Gly Gly Ala Glu Met Glu Leu TrpSer 225 230 235 309 286 PRT Oryza sativa G3378 polypeptide 309 Met HisThr Tyr Ile Tyr Thr Pro Arg Ala Ala Glu Leu Glu His Ser 1 5 10 15 HisSer Ala Ser Ala Thr Arg Ser His Ser Leu Gly Gln Ala Pro Pro 20 25 30 SerLeu Asp Arg Ser Arg Ala Ala Met Asp Met Ala Gly His Glu Val 35 40 45 AsnSer Ser Ser Ser Ser Ser Gly Ala Glu Ser Ser Ser Ser Ser Ser 50 55 60 GlyArg Gln Gln Tyr Lys Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg 65 70 75 80Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg Arg Gly Gly Ala 85 90 95Gly Arg Trp Val Cys Glu Val Arg Val Pro Gly Lys Arg Gly Ala Arg 100 105110 Leu Trp Leu Gly Thr Tyr Val Thr Ala Glu Ala Ala Ala Arg Ala His 115120 125 Asp Ala Ala Met Ile Ala Leu Arg Gly Gly Ala Gly Gly Gly Gly Ala130 135 140 Ala Cys Leu Asn Phe Gln Asp Ser Ala Trp Leu Leu Ala Val ProPro 145 150 155 160 Ala Ala Pro Ser Asp Leu Ala Gly Val Arg Arg Ala AlaThr Glu Ala 165 170 175 Val Ala Gly Phe Leu Gln Arg Asn Lys Thr Thr AsnGly Ala Ser Val 180 185 190 Ala Glu Ala Ile Asp Glu Ala Thr Ser Gly ValSer Lys Pro Pro Pro 195 200 205 Leu Ala Asn Asn Ala Asp Ser Ser Glu ThrPro Gly Pro Ser Ser Ile 210 215 220 Asp Gly Thr Ala Asp Thr Ala Ala GlyAla Ala Leu Asp Met Phe Glu 225 230 235 240 Leu Asp Phe Phe Gly Glu MetAsp Tyr Asp Thr Tyr Tyr Ala Ser Leu 245 250 255 Ala Glu Gly Leu Leu MetGlu Pro Pro Pro Ala Ala Thr Ala Leu Trp 260 265 270 Asp Asn Gly Asp GluGly Ala Asp Ile Ala Leu Trp Ser Tyr 275 280 285 310 238 PRT Oryza sativaG3379 polypeptide 310 Met Cys Gly Ile Lys Gln Glu Met Ser Gly Glu SerSer Gly Ser Pro 1 5 10 15 Cys Ser Ser Ala Ser Ala Glu Arg Gln His GlnThr Val Trp Thr Ala 20 25 30 Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys PheArg Glu Thr Arg His 35 40 45 Pro Val Phe Arg Gly Val Arg Arg Arg Gly AsnAla Gly Arg Trp Val 50 55 60 Cys Glu Val Arg Val Pro Gly Arg Arg Gly CysArg Leu Trp Leu Gly 65 70 75 80 Thr Phe Asp Thr Ala Glu Gly Ala Ala ArgAla His Asp Ala Ala Met 85 90 95 Leu Ala Ile Asn Ala Gly Gly Gly Gly GlyGly Gly Ala Cys Cys Leu 100 105 110 Asn Phe Ala Asp Ser Ala Trp Leu LeuAla Val Pro Arg Ser Tyr Arg 115 120 125 Thr Leu Ala Asp Val Arg His AlaVal Ala Glu Ala Val Glu Asp Phe 130 135 140 Phe Arg Arg Arg Leu Ala AspAsp Ala Leu Ser Ala Thr Ser Ser Ser 145 150 155 160 Ser Thr Thr Pro SerThr Pro Arg Thr Asp Asp Asp Glu Glu Ser Ala 165 170 175 Ala Thr Asp GlyAsp Glu Ser Ser Ser Pro Ala Ser Asp Leu Ala Phe 180 185 190 Glu Leu AspVal Leu Ser Asp Met Gly Trp Asp Leu Tyr Tyr Ala Ser 195 200 205 Leu AlaGln Gly Met Leu Met Glu Pro Pro Ser Ala Ala Leu Gly Asp 210 215 220 AspGly Asp Ala Ile Leu Ala Asp Val Pro Leu Trp Ser Tyr 225 230 235 311 246PRT Zea mays G3438 polypeptide 311 Met Asp Met Gly Arg His Gln Leu GlnLeu Gln His Ala Ala Ser Ser 1 5 10 15 Ser Ser Thr Ser Ala Ser Ser SerSer Glu Gln Asp Lys Pro Leu Cys 20 25 30 Cys Ser Gly Pro Lys Lys Arg ProAla Gly Arg Thr Lys Phe Arg Glu 35 40 45 Thr Arg His Pro Val Phe Arg GlyVal Arg Arg Arg Gly Ala Ala Gly 50 55 60 Arg Trp Val Cys Glu Val Arg ValPro Gly Arg Arg Gly Ala Arg Leu 65 70 75 80 Trp Leu Gly Thr Tyr Leu GlyAla Glu Ala Ala Ala Arg Ala His Asp 85 90 95 Ala Ala Met Leu Ala Leu GlyArg Gly Ala Ala Cys Leu Asn Phe Pro 100 105 110 Asp Ser Ala Trp Leu LeuAla Val Pro Pro Pro Pro Ala Leu Ser Gly 115 120 125 Gly Leu Asp Gly AlaArg Arg Ala Ala Leu Glu Ala Val Ala Glu Phe 130 135 140 Gln Arg Arg ArgPhe Gly Ala Ala Ala Ala Asp Glu Ala Thr Ser Gly 145 150 155 160 Thr SerPro Pro Ser Ser Ser Ser Ser Ala Thr Lys Pro Ala Pro Ala 165 170 175 IleGlu Arg Val Pro Val Glu Ala Ser Glu Thr Val Ala Leu Asp Gly 180 185 190Ala Val Phe Glu Pro Asp Trp Phe Gly Asp Met Asp Leu Asp Leu Tyr 195 200205 Tyr Ala Ser Leu Ala Glu Gly Leu Leu Val Glu Pro Pro Pro Pro Pro 210215 220 Pro Pro Ala Ala Trp Asp His Gly Asp Cys Cys Asp Ser Gly Ala Asp225 230 235 240 Val Ala Leu Trp Ser Tyr 245 312 256 PRT Zea mays G3439polypeptide 312 Met Asp Met Gly Arg Leu Gln Leu Gln Leu Gln His Ala AlaSer Ser 1 5 10 15 Ser Ser Thr Ser Ala Ser Ser Ser Ser Ser Ser Glu GlnAsn Lys Leu 20 25 30 Ala Trp Ser Pro Ser Ser Pro Gln Pro Pro Lys Lys ArgPro Ala Gly 35 40 45 Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val Phe ArgGly Val Arg 50 55 60 Arg Arg Gly Ala Ala Gly Arg Trp Val Cys Glu Val ArgVal Pro Gly 65 70 75 80 Arg Arg Gly Ala Arg Leu Trp Leu Gly Thr Tyr LeuGly Ala Glu Ala 85 90 95 Ala Ala Arg Ala His Asp Ala Ala Met Leu Ala LeuGly Arg Gly Ala 100 105 110 Ala Cys Leu Asn Phe Pro Asp Ser Ala Trp LeuLeu Ala Val Pro Pro 115 120 125 Pro Pro Ala Leu Ser Gly Gly Leu Asp GlyAla Arg Arg Ala Ala Leu 130 135 140 Glu Ala Val Ala Glu Phe Gln Arg ArgArg Phe Gly Ala Val Ala Ala 145 150 155 160 Asp Glu Ala Thr Ser Gly ThrSer Pro Pro Ser Ser Ser Ser Ser Pro 165 170 175 Ser Gly Thr Tyr Val SerGln Ala Pro Ala Pro Ala Ile Glu Arg Val 180 185 190 Pro Val Glu Ala SerGlu Thr Ala Ala Leu Asp Gly Ala Val Phe Glu 195 200 205 Pro Asp Trp PheArg Asp Met Asp Leu Asp Leu Tyr Tyr Ala Ser Leu 210 215 220 Ala Glu GlyLeu Leu Val Glu Pro Pro Pro Pro Pro Ala Ala Trp Asp 225 230 235 240 HisGly Asp Cys Ser His Ser Gly Ala Asp Val Ala Leu Trp Ser Tyr 245 250 255313 231 PRT Zea mays G3440 polypeptide 313 Met Cys Pro Thr Lys Lys GlyMet Thr Gly Glu Pro Ser Ser Pro Cys 1 5 10 15 Ser Ser Ala Ser Ala SerThr Leu Pro Glu His His Gln Thr Val Trp 20 25 30 Thr Ser Pro Pro Lys ArgPro Ala Gly Arg Thr Lys Phe Arg Glu Thr 35 40 45 Arg His Pro Val Phe ArgGly Val Arg Arg Arg Gly Ser Ala Gly Arg 50 55 60 Trp Val Cys Glu Val ArgVal Pro Gly Arg Arg Gly Cys Arg Leu Trp 65 70 75 80 Leu Gly Thr Phe AspThr Ala Glu Ala Ala Ala Arg Ala His Asp Ala 85 90 95 Ala Met Leu Ala LeuAla Gly Ala Gly Ala Cys Cys Leu Asn Phe Ala 100 105 110 Asp Ser Ala TrpLeu Leu Ala Val Pro Ala Ser Cys Ala Ser Leu Ala 115 120 125 Glu Val ArgHis Ala Val Ala Asp Ala Val Asp Asp Phe Leu Arg His 130 135 140 Gln LeuVal Pro Glu Asp Asp Ala Leu Ala Ala Thr Pro Ser Ser Pro 145 150 155 160Ser Ser Glu Asp Gly Asn Thr Ser Asp Gly Gly Glu Ser Ser Ser Asp 165 170175 Ser Ser Pro Pro Thr Gly Ala Ser Pro Phe Glu Phe Asp Val Phe Asn 180185 190 Asp Met Ser Trp Asp Leu His Tyr Ala Ser Leu Ala Gln Gly Leu Leu195 200 205 Val Glu Pro Pro Ser Ala Val Thr Ala Phe Met Asp Glu Gly PheAla 210 215 220 Asp Val Pro Leu Trp Ser Tyr 225 230 314 231 PRT Zea maysG3441 polypeptide 314 Met Glu Tyr Ala Ala Val Gly Tyr Gly Tyr Gly TyrGly Tyr Asp Glu 1 5 10 15 Arg Gln Glu Pro Ala Glu Ser Ala Asp Gly GlyGly Gly Gly Asp Asp 20 25 30 Glu Tyr Ala Thr Val Leu Ser Ala Pro Pro LysArg Pro Ala Gly Arg 35 40 45 Thr Lys Phe Arg Glu Thr Arg His Pro Val TyrArg Gly Val Arg Arg 50 55 60 Arg Gly Pro Ala Gly Arg Trp Val Cys Glu ValArg Glu Pro Asn Lys 65 70 75 80 Lys Ser Arg Ile Trp Leu Gly Thr Phe AlaThr Pro Glu Ala Ala Ala 85 90 95 Arg Ala His Asp Val Ala Ala Leu Ala LeuArg Gly Arg Ala Ala Cys 100 105 110 Leu Asn Phe Ala Asp Ser Ala Arg LeuLeu Gln Val Asp Pro Ala Thr 115 120 125 Leu Ala Thr Pro Asp Asp Ile ArgArg Ala Ala Ile Gln Leu Ala Asp 130 135 140 Ala Ala Ser Gln Gln Asp GluThr Ala Ala Val Ala Ala Asp Val Val 145 150 155 160 Ala Pro Ser Gln AlaAsp Asp Val Ala Ala Ala Ala Ala Ala Ala Ala 165 170 175 Ala Ala Ala MetTyr Gly Gly Gly Met Glu Phe Asp His Ser Tyr Cys 180 185 190 Tyr Asp AspGly Met Val Ser Gly Ser Ser Asp Cys Trp Gln Ser Gly 195 200 205 Gly GlyGly Trp His Ser Ser Val Asp Gly Asp Asp Asp Gly Ala Gly 210 215 220 AspMet Thr Leu Trp Ser Tyr 225 230 315 267 PRT Zea mays G3442 polypeptide315 Met Asp Thr Ala Gly Leu Val Gln His Ala Thr Ser Ser Ser Ser Thr 1 510 15 Ser Thr Ser Ala Ser Ser Ser Ser Ser Glu Gln Gln Ser Arg Lys Ala 2025 30 Ala Trp Pro Pro Ser Thr Ala Ser Ser Pro Gln Gln Pro Pro Lys Lys 3540 45 Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val Phe 5055 60 Arg Gly Val Arg Arg Arg Gly Ala Ala Gly Arg Trp Val Cys Glu Val 6570 75 80 Arg Val Pro Gly Arg Arg Gly Ala Arg Leu Trp Leu Gly Thr Tyr Leu85 90 95 Ala Ala Glu Ala Ala Ala Arg Ala His Asp Ala Ala Ile Leu Ala Leu100 105 110 Gln Gly Arg Gly Ala Gly Arg Leu Asn Phe Pro Asp Ser Ala ArgLeu 115 120 125 Leu Ala Val Pro Pro Pro Ser Ala Leu Pro Gly Leu Asp AspAla Arg 130 135 140 Arg Ala Ala Leu Glu Ala Val Ala Glu Phe Gln Arg ArgSer Gly Ser 145 150 155 160 Gly Ser Gly Ala Ala Asp Glu Ala Thr Ser GlyAla Ser Pro Pro Ser 165 170 175 Ser Ser Pro Ser Leu Pro Asp Val Ser AlaAla Gly Ser Pro Ala Ala 180 185 190 Ala Leu Glu His Val Pro Val Lys AlaAsp Glu Ala Val Ala Leu Asp 195 200 205 Leu Asp Gly Asp Val Phe Gly ProAsp Trp Phe Gly Asp Met Gly Leu 210 215 220 Glu Leu Asp Ala Tyr Tyr AlaSer Leu Ala Glu Gly Leu Leu Val Glu 225 230 235 240 Pro Pro Pro Pro ProAla Ala Trp Asp His Gly Asp Cys Cys Asp Ser 245 250 255 Gly Ala Ala AspVal Ala Leu Trp Ser Tyr Tyr 260 265 316 215 PRT Medicago sativa G3497polypeptide 316 Met Leu Thr Thr Asn Asn Ser Ser Asn Ser Gln Ser Ile SerSer Thr 1 5 10 15 Ala Ser Ser Ser Tyr Asp Met Ser Thr Pro Asn Leu GluVal Arg Leu 20 25 30 Ala Ala Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys IlePhe Lys Glu 35 40 45 Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg AsnLeu Asp Lys 50 55 60 Trp Val Cys Glu Met Arg Glu Pro Asn Thr Lys Thr ArgIle Trp Leu 65 70 75 80 Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Gln AlaHis Asp Val Ala 85 90 95 Ala Met Ala Leu Arg Gly Arg Tyr Ala Cys Val AsnPhe Ala Asp Ser 100 105 110 Val Trp Arg Leu Pro Ile Pro Ala Thr Ser LysIle Lys Asp Ile Gln 115 120 125 Lys Ala Ala Ala Glu Ala Ala Glu Ala PheArg Pro Asp Lys Thr Leu 130 135 140 Met Thr Asn Asp Ile Asp Thr Val ValAla Val Val Ala Thr Lys Glu 145 150 155 160 Leu Asn Met Phe Cys Val GluVal Glu Asp Asp Val Leu Asn Met Pro 165 170 175 Glu Leu Trp Arg Asn MetAla Leu Met Ser Arg Thr His Ser Phe Gly 180 185 190 Tyr Asp Asp Gln TyrGlu Asp Ile His Val Gln Asp Phe Gln Asp Asp 195 200 205 Glu Val Ser LeuTrp Asn Phe 210 215 317 214 PRT Medicago sativa G3498 polypeptide 317Met Phe Thr Thr Asn Asn Ser Ser Tyr Ser His Ser Ile Ser Ser Lys 1 5 1015 Ala Ser Ser Ser Tyr Tyr Asn Ser Leu Pro Asp Ser Glu Ile Arg Leu 20 2530 Ala Ala Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys Ile Phe Lys Glu 35 4045 Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Leu Asp Lys 50 5560 Trp Val Cys Glu Met Arg Glu Pro Asn Met Lys Thr Arg Ile Trp Leu 65 7075 80 Gly Thr Phe Pro Thr Ala Asp Met Ala Ala Arg Ala His Asp Val Ala 8590 95 Ala Lys Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Tyr Ser100 105 110 Val Trp Arg Leu Pro Ile Pro Ala Thr Ser Ser Ile Lys Asp IleGln 115 120 125 Lys Ala Ala Thr Lys Ala Ala Glu Ala Phe Arg Pro Asp HisThr Ile 130 135 140 Met Ile Thr Asp Ile Glu Thr Val Val Ala Val Val AlaThr Lys Asp 145 150 155 160 Leu Asn Ile Phe Cys Gly Glu Glu Glu His GluMet Leu Asp Met Ser 165 170 175 Glu Leu Trp Arg Asn Met Ala Leu Met SerPro Thr His Ser Phe Ser 180 185 190 Asn Asp His Tyr Glu Asp Ile Gln AlaGln Asp Phe Gln Asp Asp Glu 195 200 205 Val Ser Leu Trp Asn Tyr 210 318215 PRT Medicago sativa G3499 polypeptide 318 Met Lys Ser Ser Leu AspGlu Ser Ser Tyr Val Glu Asn Asn Ser Ser 1 5 10 15 Ser Ser Glu Thr SerCys Tyr Glu Glu Ile Leu Leu Ala Ser Glu Arg 20 25 30 Pro Lys Lys Pro AlaGly Arg Arg Lys Phe Lys Glu Thr Arg His Pro 35 40 45 Val Tyr Arg Gly ValArg Arg Arg Asn Asn Asn Lys Trp Val Cys Glu 50 55 60 Val Arg Val Pro AsnAsp Lys Ser Thr Arg Ile Trp Leu Gly Thr Tyr 65 70 75 80 Pro Thr Pro GluMet Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 85 90 95 Leu Arg Gly LysSer Ala Cys Leu Asn Phe Ala Asn Ser Ala Trp Arg 100 105 110 Leu Ala LeuPro Glu Thr Asn Asn Ala Lys Glu Ile Arg Lys Met Ala 115 120 125 Ala GluAla Ala Leu Ala Phe Ala Val Glu Asp Ser Lys Glu Gln Ile 130 135 140 MetIle Ser Asn Cys Asp Val Cys Ser Ser Asp Asn Val Met Glu Val 145 150 155160 Asp Asn Lys Pro Leu Gln Gly Leu Cys Val Glu Val Pro Glu Gln Glu 165170 175 Met Leu His Asp Trp Phe Arg Ser Met Ala Asp Glu Pro Leu Arg Ser180 185 190 Pro Met Thr Pro Phe Ile Arg Tyr Gly Ile Gly Arg Asp His SerAsn 195 200 205 Asn Val Asp Val Asp Pro Cys 210 215 319 7 PRT Artificialsequence oligopeptide 319 Pro Lys Xaa Xaa Ala Gly Arg 1 5 320 6 PRTArtificial sequence oligopeptide 320 Ala Gly Arg Xaa Lys Phe 1 5 321 5PRT oligopeptide oligopeptide 321 Glu Thr Arg His Pro 1 5 322 7 PRTArtificial sequence oligopeptide 322 Pro Lys Lys Xaa Ala Gly Arg 1 5 3237 PRT Artificial sequence oligopeptide 323 Pro Lys Lys Arg Ala Gly Arg 15 324 6 PRT Artificial sequence oligopeptide 324 Ala Gly Arg Lys Xaa Phe1 5 325 6 PRT Artificial sequence oligopeptide 325 Ala Gly Arg Lys LysPhe 1 5 326 7 PRT Artificial sequence oligopeptide 326 Pro Lys Lys ProAla Gly Arg 1 5 327 79 PRT Arabidopsis thaliana CBF1 AP2 domain andflanking signature sequences 327 Pro Lys Lys Pro Ala Gly Arg Lys Lys PheArg Glu Thr Arg His Pro 1 5 10 15 Ile Tyr Arg Gly Val Arg Gln Arg AsnSer Gly Lys Trp Val Ser Glu 20 25 30 Val Arg Glu Pro Asn Lys Lys Thr ArgIle Trp Leu Gly Thr Phe Gln 35 40 45 Thr Ala Glu Met Ala Ala Arg Ala HisAsp Val Ala Ala Leu Ala Leu 50 55 60 Arg Gly Arg Ser Ala Cys Leu Asn PheAla Asp Ser Ala Trp Arg 65 70 75 328 79 PRT Arabidopsis thaliana CBF2AP2 domain and flanking signature sequences 328 Pro Lys Lys Pro Ala GlyArg Lys Lys Phe Arg Glu Thr Arg His Pro 1 5 10 15 Ile Tyr Arg Gly ValArg Gln Arg Asn Ser Gly Lys Trp Val Cys Glu 20 25 30 Leu Arg Glu Pro AsnLys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 35 40 45 Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala Ile Ala Leu 50 55 60 Arg Gly Arg Ser AlaCys Leu Asn Phe Ala Asp Ser Ala Trp Arg 65 70 75 329 79 PRT Arabidopsisthaliana CBF3 AP2 domain and flanking signature sequences 329 Pro LysLys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro 1 5 10 15 IleTyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val Cys Glu 20 25 30 ValArg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 35 40 45 ThrAla Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 50 55 60 ArgGly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 65 70 75 330 79PRT Arabidopsis thaliana G912 AP2 domain and flanking signaturesequences 330 Pro Lys Lys Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr ArgHis Pro 1 5 10 15 Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys TrpVal Cys Glu 20 25 30 Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu GlyThr Phe Pro 35 40 45 Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala AlaLeu Ala Leu 50 55 60 Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser AlaTrp Arg 65 70 75 331 210 PRT Lycopersicon esculentum Lycopersiconesculentum CBF1 polypeptide 331 Met Asn Ile Phe Glu Thr Tyr Tyr Ser AspSer Leu Ile Leu Thr Glu 1 5 10 15 Ser Ser Ser Ser Ser Ser Ser Ser SerPhe Ser Glu Glu Glu Val Ile 20 25 30 Leu Ala Ser Asn Asn Pro Lys Lys ProAla Gly Arg Lys Lys Phe Arg 35 40 45 Glu Thr Arg His Pro Ile Tyr Arg GlyIle Arg Lys Arg Asn Ser Gly 50 55 60 Lys Trp Val Cys Glu Val Arg Glu ProAsn Lys Lys Thr Arg Ile Trp 65 70 75 80 Leu Gly Thr Phe Pro Thr Ala GluMet Ala Ala Arg Ala His Asp Val 85 90 95 Ala Ala Leu Ala Leu Arg Gly ArgSer Ala Cys Leu Asn Phe Ser Asp 100 105 110 Ser Ala Trp Arg Leu Pro IlePro Ala Ser Ser Asn Ser Lys Asp Ile 115 120 125 Gln Lys Ala Ala Ala GlnAla Val Glu Ile Phe Arg Ser Glu Glu Val 130 135 140 Ser Gly Glu Ser ProGlu Thr Ser Glu Asn Val Gln Glu Ser Ser Asp 145 150 155 160 Phe Val AspGlu Glu Ala Ile Phe Phe Met Pro Gly Leu Leu Ala Asn 165 170 175 Met AlaGlu Gly Leu Met Leu Pro Pro Pro Gln Cys Ala Glu Met Gly 180 185 190 AspHis Cys Val Glu Thr Asp Ala Tyr Met Ile Thr Leu Trp Asn Tyr 195 200 205Ser Ile 210 332 220 PRT Lycopersicon esculentum Lycopersicon esculentumCBF2 polypeptide 332 Met Asp Ile Phe Glu Ser Tyr Tyr Ser Asn Ser Phe ValGlu Ser Leu 1 5 10 15 Leu Ser Ser Ser Leu Ser Ile Ser Asp Thr Asn AsnLeu Asn His Tyr 20 25 30 Ser Pro Asn Glu Glu Val Ile Ile Leu Ala Ser AsnAsn Pro Lys Lys 35 40 45 Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg HisPro Val Tyr Arg 50 55 60 Gly Ile Arg Lys Arg Asn Ser Gly Lys Trp Val CysGlu Val Arg Glu 65 70 75 80 Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly ThrPhe Pro Thr Ala Glu 85 90 95 Met Ala Ala Arg Ala His Asp Val Ala Ala IleAla Leu Arg Gly Arg 100 105 110 Ser Ala Cys Leu Asn Phe Ala Asp Ser TyrTrp Arg Leu Pro Ile Pro 115 120 125 Ala Ser Ser Asn Ser Lys Asp Ile GlnLys Ala Ala Ala Glu Ala Ala 130 135 140 Glu Ile Phe Arg Ser Glu Glu ValSer Gly Glu Ser Pro Glu Thr Ser 145 150 155 160 Glu Asn Val Gln Glu SerSer Asp Phe Val Asp Glu Glu Ala Leu Phe 165 170 175 Ser Met Pro Gly LeuLeu Ala Asn Met Ala Glu Gly Leu Met Leu Pro 180 185 190 Pro Pro Gln CysLeu Glu Ile Gly Asp His Tyr Val Glu Leu Ala Asp 195 200 205 Val His AlaTyr Met Pro Leu Trp Asn Tyr Ser Ile 210 215 220

What is claimed is:
 1. A transgenic plant having an altered trait ascompared to a wild-type or untransformed plant, wherein said alteredtrait is selected from the group consisting of: (a) altered levels of atleast one cell protectant in cells of said transgenic plant; and (b)altered biomass; wherein said transgenic plant is transformed with arecombinant polynucleotide comprising a nucleotide sequence that encodesa CBF, and said CBF comprises an AP2 domain; wherein the altered levelsof at least one cell protectant is selected from the group consisting of(i) increasing; and (ii) decreasing levels; and wherein the alteredbiomass is selected from the group consisting of: (i) increasing; and(ii) decreasing biomass.
 2. The transgenic plant of claim 1, whereinsaid nucleotide sequence is selected from the group consisting of SEQ IDNO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115,117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.3. The transgenic plant of claim 1, wherein said CBF is selected fromthe group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116,118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, andfragments thereof.
 4. The transgenic plant of claim 1, wherein saidnucleotide sequence specifically hybridizes under highly stringentconditions to SEQ ID NO: 1 or its complement under the conditions of6×SSC and 65° C.
 5. The transgenic plant of claim 1, wherein expressionof said CBF modifies the level of said at least one cell protectant insaid cells of said transgenic plant.
 6. The transgenic plant of claim 1,wherein said transgenic plant is a crop plant selected from the groupconsisting of Brassica juncea, Brassica napus, Brassica oleracea,Brassica rapa, Brassica rapa L, Brassica napus L, Glycine max, Raphanussativus, Zea mays, Triticum, Oryza sativa, Secale cereale, Sorghumbicolor, Sorghum vulgare, and Hordeum vulgare.
 7. The transgenic plantof claim 1, wherein said transgenic plant has increased tolerance to anenvironmental stress.
 8. The transgenic plant of claim 7, wherein saidenvironmental stress is selected from the group consisting of drought,cold, freezing, and high salt.
 9. A transgenic plant having an alteredtrait as compared to a wild-type or untransformed plant, wherein saidaltered trait is selected from the group consisting of: (a) alteredlevels of at least one cell protectant in cells of said transgenicplant; and (b) altered biomass; wherein said transgenic plant istransformed with a recombinant polynucleotide comprising a nucleotidesequence that encodes a CBF, and said CBF comprises a sequence selectedfrom the group consisting of: (a) an AP2 domain comprising consecutiveamino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQID NO: 325; or (d) SEQ ID NO: 321; wherein amino acids 45, 46, 48,50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91,93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2domain of the CBF; and wherein the alteration in levels of the at leastone cell protectant is selected from the group consisting of (i)increasing; and (ii) decreasing levels; and wherein the altered biomassis selected from the group consisting of (i) increasing; and (ii)decreasing biomass.
 10. The transgenic plant of claim 9, wherein saidnucleotide sequence is selected from the group consisting of SEQ ID NO:1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117,119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof. 11.The transgenic plant of claim 9, wherein said CBF is selected from thegroup consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116,118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, andfragments thereof
 12. A transgenic rapeseed plant having altered biomassas compared to a wild-type or untransformed plant, wherein saidtransgenic rapeseed plant is transformed with a recombinantpolynucleotide comprising a nucleotide sequence that encodes a CBFcomprising an AP2 domain; and wherein the altered biomass is selectedfrom the group consisting of (i) increasing; and (ii) decreasingbiomass.
 13. The transgenic rapeseed plant of claim 12, wherein saidnucleotide sequence is selected from the group consisting of SEQ ID NO:1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117,119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof. 14.The transgenic rapeseed plant of claim 12, wherein said CBF is selectedfrom the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, andfragments thereof
 15. The transgenic rapeseed plant of claim 12, whereinsaid nucleotide sequence specifically hybridizes under highly stringentconditions to SEQ ID NO: 1 or its complement under the conditions of6×SSC and 65° C.
 16. The transgenic rapeseed plant of claim 12, whereinsaid nucleotide sequence further comprises a regulatory region operablylinked to the sequence encoding the CBF.
 17. The transgenic rapeseedplant of claim 16, wherein said regulatory region is a constitutivepromoter, an inducible promoter, a tissue specific promoter, or adevelopmental stage specific promoter.
 18. A transgenic rapeseed planthaving altered biomass, said transgenic rapeseed plant comprising arecombinant polynucleotide comprising a nucleotide sequence encoding aCBF comprises a sequence selected from the group consisting of: (a) anAP2 domain comprising consecutive amino acid residues 45-106 of SEQ IDNO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321;wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65,67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 ofSEQ ID NO:2 are conserved in the AP2 domain of the CBF; and wherein saidbiomass is altered compared to that of a plant that has not beentransformed with said recombinant polynucleotide; and wherein thealtered biomass is selected from the group consisting of (i) increasing;and (ii) decreasing biomass.
 19. A transgenic rapeseed plant havingincreased environmental stress tolerance, said transgenic rapeseed plantcomprising a recombinant polynucleotide comprising a nucleotide sequenceencoding a CBF comprising an AP2 domain.
 20. The transgenic rapeseedplant of claim 19, wherein said nucleotide sequence is selected from thegroup consisting of SEQ ID NO 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs,and variants thereof.
 21. The transgenic rapeseed plant of claim 19,wherein said CBF is selected from the group consisting of SEQ ID NO: 2,10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128,orthologs, paralogs, variants, and fragments thereof.
 22. The transgenicrapeseed plant of claim 19, wherein said nucleotide sequencespecifically hybridizes under highly stringent conditions to SEQ ID NO:1 or its complement under the conditions of 6×SSC and 65° C.
 23. Thetransgenic rapeseed plant of claim 19, wherein said CBF comprises asequence selected from the group consisting of: (a) an AP2 domaincomprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b)SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein aminoacid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73,75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2are conserved in the AP2 domain of the CBF.
 24. The transgenic rapeseedplant of claim 19, wherein said recombinant polynucleotide furthercomprises a regulatory region operably linked to the sequence encodingthe CBF.
 25. The transgenic rapeseed plant of claim 24, wherein saidregulatory region is a constitutive promoter, an inducible promoter, atissue specific promoter or a developmental stage specific promoter. 26.The transgenic rapeseed plant of claim 19, wherein said environmentalstress is selected from the group consisting of dehydration stress, coldstress, and freezing stress.
 27. A cold-inducible promoter comprising anucleotide sequence selected from the group consisting of nucleotides1-1695 of SEQ ID NO: 129 and nucleotides 1-1952 of SEQ ID NO:
 130. 28.The cold-inducible promoter of claim 27, wherein said nucleotidesequence is selected from the group consisting of nucleotides 1470-1520of SEQ ID NO: 129 and nucleotides 460-520 of SEQ ID NO:
 130. 29. Amethod for modifying the levels of a cell protectant in a cell, saidmethod comprising: (a) transforming the cell with a recombinantpolynucleotide comprising a nucleotide sequence encoding a CBF; and (b)expressing said CBF in the transformed cell; wherein said transformedcell has altered cell protectant levels compared with a cell that hasnot been transformed with said recombinant polynucleotide.
 30. Themethod of claim 29, wherein said nucleotide sequence is selected fromthe group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs,paralogs, and variants thereof.
 31. The method of claim 29, wherein saidCBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs,variants, and fragments thereof
 32. The method of claim 29, wherein saidnucleotide sequence specifically hybridizes under highly stringentconditions to SEQ ID NO: 1 or its complement under the conditions of6×SSC and 65° C.
 33. The method of claim 29, wherein said cellprotectant is selected from the group consisting of proline, sucrose,and a fatty acid.
 34. The method of claim 29, wherein said cellprotectant is a fatty acid selected from the group consisting of 16:1,16:2, or 18:0, and 18:1 fatty acids.
 35. The method of claim 29, whereinsaid CBF binds to a member of a class of DNA regulatory sequences thatincludes a subsequence selected from the group consisting of CCGAA,CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG,CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG,TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG,GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA,CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
 36. The method ofclaim 29, wherein said CBF binds to a cold or dehydrationtranscription-regulating region comprising the sequence CCG.
 37. Themethod of claim 29, wherein said CBF comprises a sequence selected fromthe group consisting of: (a) an AP2 domain comprising consecutive aminoacid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO:325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48,50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91,93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2domain of the CBF.
 38. The method of claim 29, wherein said CBF is CBF3.39. The method of claim 29, further comprising cold-acclimating saidtransformed cell.
 40. The method of claim 29, wherein said recombinantpolynucleotide further comprises a regulatory region operably linked tothe sequence encoding the CBF.
 41. The method of claim 40, wherein saidregulatory region is a constitutive promoter, an inducible promoter, atissue specific promoter, or a developmental stage specific promoter.42. The method of claim 29, wherein said CBF comprises an amino acidsequence homologous to a sequence selected from an amino acid sequencedepicted in FIG. 19A, 19B, 19C, 19D, or 19E that binds to a DNAregulatory sequence that induces expression of an environmental stresstolerance gene and modifies cell protectant levels.
 43. The method ofclaim 29, wherein the amino acid sequence comprises consecutive aminoacid residues of Thr-Xaa₍₁₃₎-Ala-Xaa₍₁₂₎-Ser, wherein Xaa represents anyamino acid residue.
 44. The method of claim 29, wherein the amino acidsequence comprises consecutive amino acid residues ofAsn-Xaa₍₁₂₎-Thr-Xaa₍₁₃₎-Ala-Leu-Arg-Xaa₍₈₎-Ala-Xaa-Ser, wherein Xaarepresents any amino acid residue.
 45. The method of claim 29, whereinthe amino acid sequence comprises consecutive amino acid residues ofGly-Val-Arg-Xaa-Arg-Tyr-Xaa₍₄₋₅₎-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa₍₆₎-Arg-Glu-Xaa-Asn-Lys-Xaa₍₂₎-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa₍₅₎-Ala-Ala-Xaa-Ala-Xaa-Asp-Xaa-Ala-Ala-Xaa₍₄₎-Gly-Xaa₍₂₎-Ala-Xaa-Leu-Asn,wherein Xaa represents any amino acid residue.
 46. The method of claim29, wherein the amino acid sequence comprises consecutive amino acidresidues ofGly-Val-Arg-Xaa-Arg-Tyr-Xaa₍₄₋₅₎-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa₍₆₎-Arg-Glu-Xaa-Asn-Lys-Xaa₍₂₎-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa-Thr-Xaa₍₃₎-Ala-Ala-Xaa-Ala-Xaa-Asp-Xaa-Ala-Ala-Xaa-Ala-Xaa₍₂₎-Gly-Xaa₍₂₎-Ala-Xaa-Leu-Asn-Xaa₍₃₎-Ser,wherein Xaa represents any amino acid residue.
 47. The method of claim29, wherein the amino acid sequence comprises consecutive amino acidresidues ofHis-Pro-Xaa-Tyr-Gly-Val-Arg-Xaa-Arg-Tyr-Xaa₍₄₋₅₎-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa-Asn-Lys-Xaa₍₂₎-Arg-Glu-Xaa-Asn-Lys-Xaa₍₂₎-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa-Thr-Xaa-Glu-Xaa-Ala-Ala-Arg-Ala-Asp-His-Asp-Val-Ala-Ala-Xaa-Ala-Leu-Arg-Gly-Xaa₍₂₎-Ala-Xaa-Leu-Asn-Xaa-Ala-Asp-Ser,wherein Xaa represents any amino acid residue.
 48. The method of claim29, wherein said recombinant polynucleotide encodes a polypeptide thatelevates cold-regulated gene levels in the absence of cold acclimationcompared with cold-regulated gene levels in a plant lacking saidrecombinant polynucleotide.
 49. A method for modifying the levels of acell protectant in a plant cell, said method comprising: (a)transforming the plant cell with a recombinant polynucleotide comprisinga nucleotide sequence encoding a CBF; wherein said CBF is sufficientlyhomologous to a consensus sequence shown in depicted in FIG. 19A, 19B,19C, 19D, or 19E that the CBF is capable of binding to a CCG regulatorysequence and that the nucleotide sequence that encodes the CBF canhybridize to SEQ ID NO: 1 under the conditions of 6×SSC and 65° C.; and(b) expressing said CBF in the transformed plant cell; wherein saidtransformed plant cell has altered cell protectant levels compared witha plant cell that has not been transformed with said recombinantpolynucleotide.
 50. A method for modifying proline, sucrose or fattyacid levels in a plant cell, said method comprising: a) transforming theplant cell with a recombinant polynucleotide comprising a nucleotidesequence encoding a CBF; and (b) expressing said CBF in the transformedcell; whereby expression of said CBF modifies proline levels in theplant cell.
 51. The method of claim 50, wherein said nucleotide sequenceis selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125,127, orthologs, paralogs, and variants thereof.
 52. The method of claim50, wherein said CBF is selected from the group consisting of SEQ ID NO:2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128,orthologs, paralogs, variants, and fragments thereof.
 53. The method ofclaim 50, wherein said nucleotide sequence specifically hybridizes underhighly stringent conditions to SEQ ID NO: 1 or its complement under theconditions of 6×SSC and 65° C.
 54. The method of claim 50, wherein saidCBF comprises a sequence selected from the group consisting of: (a) anAP2 domain comprising consecutive amino acid residues 45-106 of SEQ IDNO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321;wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65,67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 ofSEQ ID NO:2 are conserved in the AP2 domain of the CBF.
 55. The methodof claim 50, wherein said CBF is CBF3.
 56. The method of claim 50,wherein said recombinant polynucleotide further comprises a regulatoryregion operably linked to the sequence encoding the CBF.
 57. The methodof claim 56, wherein said regulatory region is a constitutive promoter,an inducible promoter, a tissue specific promoter, or a developmentalstage specific promoter.
 58. A method for improving the tolerance of aplant cell to an environmental stress, said method comprising (a)transforming the plant cell with a recombinant polynucleotide comprisinga nucleotide sequence encoding a CBF; wherein said CBF is sufficientlyhomologous to a consensus sequence shown in depicted in FIG. 19A, 19B,19C, 19D, or 19E that the CBF is capable of binding to a CCG regulatorysequence and that the nucleotide sequence that encodes the CBF canhybridize to SEQ ID NO: 1 under highly stringent conditions of 6×SSC and65° C.; and (b) expressing said CBF in the transformed plant cell;whereby expression of said CBF increases cell protectant levels at least1.5 fold in the transformed plant cell compared with cell protectantlevels in an untransformed plant cell and the increased cell protectantlevels in the transformed plant cell improve the tolerance of thetransformed plant cell to the environmental stress, and wherein theenvironmental stress is selected from the group consisting of: cold,freezing, dehydration, and drought; and wherein the improvedenvironmental stress tolerance is selected from the group consisting of:a) a decrease in the extent of a plant's injury; b) a decrease in theextent of a cell's injury; c) a decrease in the extent of a plant'sgrowth inhibition; d) a decrease in the extent of a cell's growthinhibition; e) an increase in survival rate after exposure to coldtemperatures; f) an increase in survival rate after exposure to freezingtemperatures; g) an increase in the extent of survival of a plant afterexposure to drought conditions; h) an increase in the extent of survivalof a cell after exposure to drought conditions; i) an increase in theextent of survival of a plant after exposure to high salt conditions;and j) an increase in the extent of survival of a cell after exposure tohigh salt conditions.
 59. The method of claim 58, wherein saidnucleotide sequence is selected from the group consisting of SEQ ID NO:1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117,119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof. 60.The method of claim 58, wherein said CBF is selected from the groupconsisting SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122,124, 126, 128, orthologs, paralogs, variants, and fragments thereof. 61.The method of claim 58, wherein said nucleotide sequence specificallyhybridizes under highly stringent conditions to SEQ ID NO: 1 or itscomplement under the conditions of 6×SSC and 65° C.
 62. The method ofclaim 58, wherein said cell protectant is selected from the groupconsisting of proline and sucrose.
 63. The method of claim 58, whereinsaid CBF comprises a sequence selected from the group consisting of: (a)an AP2 domain comprising consecutive amino acid residues 45-106 of SEQID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321;wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65,67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 ofSEQ ID NO:2 are conserved in the AP2 domain of the CBF.
 64. The methodof claim 58, wherein said CBF is CBF3.
 65. The method of claim 58,wherein said recombinant polynucleotide further comprises a regulatoryregion operably linked to the sequence encoding the CBF.
 66. The methodof claim 65, wherein said regulatory region is a constitutive promoter,an inducible promoter, a tissue specific promoter or a developmentalstage specific promoter.
 67. The method of claim 58, further comprisingcold-acclimating said transformed cell.
 68. A method of screening for aCBF, said method comprising: (a) constructing yeast reporter strains bytransforming yeast cells with a vector comprising C-repeat/DRE sequencesas upstream activator sequence elements fused upstream of a reportergene; (b) transforming said yeast reporter strains with an expressionlibrary containing random cDNA inserts each of which are fused to anactivation domain; (c) growing said yeast reporter strains ondifferential media; and (c) screening for yeast colonies that comprisecDNA inserts encoding C-repeat/DRE binding domains fused to saidactivation domains.
 69. The method of claim 68, wherein said reportergene is a lacZ reporter gene, said reporter gene is operably linked to aGAL1 promoter, and said activation domain is a GAL4 activation domain;wherein clones that contain a cDNA insert encoding a C-repeat/DREbinding domain fused to GAL4-activation domains bind upstream of thelacZ reporter gene carrying the wild type C-repeat/DRE sequence,activating transcription of the lacZ reporter gene. and wherein saiddifferential media comprises X-gal treated filters, and said yeastcolonies on said X-gal-treated filters are screened by their blue color.70. The method of clain 68, wherein said CBF binds to a member of aclass of DNA regulatory sequences that includes a subsequence selectedfrom the group consisting of CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT,CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG,AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG,CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG,TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT,GCCGC, and GCCGG.
 71. A method for identifying a CBF, said methodcomprising: (a) identifying sequences by BLAST analysis in which ESTs ina database are queried using SEQ ID NO: 327, 328, 329, 330, or 331 asthe query sequence (b) selecting positive EST sequences with an HSPBLAST score of at least 100 bits over at least 95% of the querysequence; and (c) organizing said positive EST sequences into contigs;thereby identifying a CBF sequence.
 72. The method of claim 71, furthercomprising: identifying complete predicted coding regions with a startand a stop codon; and cloning said CBF sequence directly from cDNA orgenomic DNA by PCR using primers in the 5′ and 3′ flanking regions. 73.The method of claim 71, further comprising cloning said CBF sequenceinto a transformation vector.
 74. The method of claim 73, wherein saidtransformation vector is pMEN65 or a transformation vector modified frompMEN65.
 75. A method for enhancing the cold tolerance of a plant, saidmethod comprising: (a) transforming a plant with a recombinantpolynucleotide comprising a nucleotide sequence encoding a CBF; (b)expressing the recombinant polynucleotide and (c) cold acclimating saidtransformed plant; wherein said cold acclimated transformed plant has animproved cold tolerance compared with a non-transformed plant that hasbeen cold-acclimated; and wherein the enhanced cold tolerance isselected from the group consisting of: a) a decrease in the extent of aplant's injury; b) a decrease in the extent of a cell's injury; c) adecrease in the extent of a plant's growth inhibition; d) a decrease inthe extent of a cell's growth inhibition; e) an increase in survivalrate after exposure to cold temperatures; and f) an increase in survivalrate after exposure to freezing temperatures.
 76. The method of claim75, wherein said plant is a rapeseed plant.
 77. The method of claim 75,wherein said polynucleotide comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127,orthologs, paralogs, and variants thereof.
 78. The method of claim 75,wherein said CBF is selected from the group consisting SEQ ID NO: 2, 10,13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs,paralogs, variants, and fragments thereof.
 79. The method of claim 75,wherein said nucleotide sequence specifically hybridizes under highlystringent conditions to SEQ ID NO: 1 or its complement under theconditions of 6×SSC and 65° C.
 80. The method of claim 75, wherein saidCBF comprises a sequence selected from the group consisting of: (a) anAP2 domain comprising consecutive amino acid residues 45-106 of SEQ IDNO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321;wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65,67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 ofSEQ ID NO:2 are conserved in the AP2 domain of the CBF.
 81. The methodof claim 75, wherein the cold tolerance is enhanced by at least 2° C.compared with the cold tolerance of the non-transformed plant that hasbeen cold-acclimated.
 82. The method of claim 75, wherein the coldtolerance is enhanced by at least 5° C. compared with the cold toleranceof the non-transformed plant that has been cold-acclimated.
 83. Themethod of claim 75, wherein the cold tolerance is enhanced by at least8° C. compared with the cold tolerance of the non-transformed plant thathas been cold-acclimated.
 84. The method of claim 75, wherein saidrecombinant polynucleotide further comprises a regulatory regionoperably linked to the sequence encoding the CBF.
 85. The method ofclaim 84, wherein said regulatory region is a constitutive promoter, aninducible promoter, a tissue specific promoter, or a developmental stagespecific promoter.
 86. The method of claim 85, wherein said induciblepromoter is a cold-inducible promoter comprising a sequence selectedfrom the group consisting of a sequence comprising nucleotides 1470-1520of SEQ ID NO: 129 and a sequence comprising nucleotides 460-520 of SEQID NO:
 130. 87. A method for altering the biomass of a plant, saidmethod comprising: (a) transforming said plant with a recombinantpolynucleotide comprising a nucleotide sequence encoding a CBF; and (b)expressing said CBF in said transformed plant; wherein said transformedplant has a altered biomass compared with a plant that has not beentransformed with said recombinant polynucleotide.
 88. The method ofclaim 87, wherein said nucleotide sequence is selected from the groupconsisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, andvariants thereof.
 89. The method of claim 87, wherein said CBF isselected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs,variants, and fragments thereof.
 90. The method of claim 87, whereinsaid nucleotide sequence specifically hybridizes under highly stringentconditions to SEQ ID NO: 1 or its complement under the conditions of6×SSC and 65° C.
 91. The method of claim 87, wherein said alteredbiomass is an increase in the leaf number, leaf size, or root mass of aplant.
 92. The method of claim 87, wherein said CBF comprises a sequenceselected from the group consisting of: (a) an AP2 domain comprisingconsecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO:326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acidresidues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73,75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO:2 areconserved in the AP2 domain of the CBF.
 93. The method of 87, whereinsaid CBF binds to a cold or dehydration transcription-regulating regioncomprising the sequence CCG.
 94. The method of claim 87, wherein saidCBF binds to a member of a class of DNA regulatory sequences whichincludes a subsequence selected from the group consisting of CCGAA,CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG,CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG,TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG,GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA,CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
 95. The method ofclaim 87, wherein said recombinant polynucleotide further comprises aregulatory region operably linked to the sequence encoding the CBF. 96.The method of claim 95, wherein said regulatory region is a constitutivepromoter, an inducible promoter, a tissue specific promoter, or adevelopmental stage specific promoter.
 97. The method of claim 87,wherein said plant is exposed to adverse environmental conditions, andsaid biomass is increased relative to an untransformed plant exposed tothe same conditions.
 98. The method of claim 87, wherein said plant cellis derived from a crop plant selected from the group consisting ofBrassica juncea, Brassica napus, Brassica oleracea, Brassica rapa,Brassica rapa L, Brassica napus L, Glycine max, Raphanus sativus, Zeamays, Triticum, Oryza sativa, Secale cereale, Sorghum bicolor, Sorghumvulgare, and Hordeum vulgare.
 99. A method for altering the biomass of aplant, said method comprising: (a) transforming said plant with arecombinant polynucleotide comprising a nucleotide sequence encoding aCBF; wherein said CBF is sufficiently homologous to a consensus sequenceshown in depicted in FIG. 19A, 19B, 19C, 19D, or 19E that the CBF iscapable of binding to a CCG regulatory sequence and that the nucleotidesequence that encodes the CBF can hybridize to SEQ ID NO: 1 under theconditions of 6×SSC and 65° C.; and (b) expressing said CBF in saidtransformed plant; wherein said transformed plant has a altered biomasscompared with a plant that has not been transformed with saidrecombinant polynucleotide.
 100. A method for altering the biomass of aplant, said method comprising: (a) altering the levels of apolynucleotide comprising a nucleotide sequence encoding a CBF in aplant; and (b) identifying a plant so altered; wherein said alteredlevels of said polynucleotide modifies the biomass of the plant comparedto a plant that does not comprise said altered polynucleotide levels.101. The method of claim 100, wherein said nucleotide sequence isselected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127,orthologs, paralogs, and variants thereof.
 102. The method of claim 100,wherein said CBF is selected from the group consisting of SEQ ID NO: 2,10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128,orthologs, paralogs, variants, and fragments thereof.
 103. The method ofclaim 100, wherein said nucleotide sequence specifically hybridizesunder highly stringent conditions to SEQ ID NO: 1 or its complementunder the conditions of 6×SSC and 65° C.
 104. The method of claim 100,wherein said altered biomass is an increase in leaf number, leaf size,or root mass.
 105. The method of claim 100, wherein said CBF comprises asequence selected from the group consisting of: (a) an AP2 domaincomprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b)SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein aminoacid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73,75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO:2 areconserved in the AP2 domain of the CBF.
 106. The method of claim 100,wherein said CBF binds to a cold or dehydration transcription-regulatingregion comprising the sequence CCG.
 107. The method of claim 100,wherein said CBF binds to a member of a class of DNA regulatorysequences that includes a subsequence selected from the group consistingof CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT,CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG,TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG,GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG,CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
 108. Themethod of claim 100, wherein said recombinant polynucleotide furthercomprises a regulatory region operably linked to the sequence encodingthe CBF.
 109. The method of claim 108, wherein said regulatory region isa constitutive promoter, an inducible promoter, a tissue specificpromoter, or a developmental stage specific promoter.
 110. The method ofclaim 100, wherein said plant is exposed to adverse environmentalconditions, and said biomass is increased relative to an untransformedplant exposed to the same conditions.
 111. The method of claim 100,wherein said plant cell is derived from a crop plant selected from thegroup consisting of Brassica juncea, Brassica napus, Brassica oleracea,Brassica rapa, Brassica rapa L, Brassica napus L, Glycine max, Raphanussativus, Zea mays, Triticum, Oryza sativa, Secale cereale, Sorghumbicolor, Sorghum vulgare, and Hordeum vulgare.